Trifluoromethyl pyrazolyl guanidine f1f0-atpase inhibitors and therapeutic uses thereof

ABSTRACT

The invention provides trifluoromethyl pyrazolyl guanidine compounds that inhibit F 1 F 0 -ATPase, and methods of using trifluoromethyl pyrazolyl guanidine compounds as therapeutic agents to treat medical disorders, such as an immune disorder, inflammatory condition, or cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. ProvisionalPatent Application Ser. No. 61/914,085, filed Dec. 10, 2013, and U.S.Provisional Patent Application Ser. No. 61/979,619, filed Apr. 15, 2014;the contents of each of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The invention provides inhibitors of F₁F₀-ATPases (e.g., mitochondrialF₁F₀-ATPases) and their therapeutic use. In particular, the inventionprovides trifluoromethyl pyrazolyl guanidine compounds that inhibitF₁F₀-ATPase, and methods of using trifluoromethyl pyrazolyl guanidinecompounds as therapeutic agents to treat a number of medical conditions.

BACKGROUND

Multicellular organisms exert precise control over cell number. Abalance between cell proliferation and cell death achieves thishomeostasis. Cell death occurs in nearly every type of vertebrate cellvia necrosis or through a suicidal form of cell death, known asapoptosis. Apoptosis is triggered by a variety of extracellular andintracellular signals that engage a common, genetically programmed deathmechanism.

Multicellular organisms use apoptosis to instruct damaged or unnecessarycells to destroy themselves for the good of the organism. Control of theapoptotic process therefore is very important to normal development, forexample, fetal development of fingers and toes requires the controlledremoval, by apoptosis, of excess interconnecting tissues, as does theformation of neural synapses within the brain. Similarly, controlledapoptosis is responsible for the sloughing off of the inner lining ofthe uterus (the endometrium) at the start of menstruation. Whileapoptosis plays an important role in tissue sculpting and normalcellular maintenance, it is also a component of the primary defenseagainst cells and invaders (e.g., viruses) which threaten the well beingof the organism.

Many diseases are associated with dysregulation of apoptotic cell death.Experimental models have established a cause-effect relationship betweenaberrant apoptotic regulation and the pathogenicity of variousneoplastic, autoimmune and viral diseases. For instance, in thecell-mediated immune response, effector cells (e.g., cytotoxic Tlymphocytes “CTLs”) destroy virus-infected cells by inducing theinfected cells to undergo apoptosis. The organism subsequently relies onthe apoptotic process to destroy the effector cells when they are nolonger needed. Autoimmunity is normally prevented by the CTLs inducingapoptosis in each other and even in themselves. Defects in this processare associated with a variety of immune diseases such as lupuserythematosus and rheumatoid arthritis.

Multicellular organisms also use apoptosis to instruct cells withdamaged nucleic acids (e.g., DNA) to destroy themselves prior tobecoming cancerous. Some cancer-causing viruses overcome this safeguardby reprogramming infected (transformed) cells to abort the normalapoptotic process. For example, several human papilloma viruses (HPVs)have been implicated in causing cervical cancer by suppressing theapoptotic removal of transformed cells by producing a protein (E6) whichinactivates the p53 apoptosis promoter. Similarly, the Epstein-Barrvirus (EBV), the causative agent of mononucleosis and Burkitt'slymphoma, reprograms infected cells to produce proteins that preventnormal apoptotic removal of the aberrant cells thus allowing thecancerous cells to proliferate and to spread throughout the organism.

Still other viruses destructively manipulate a cell's apoptoticmachinery without directly resulting in the development of a cancer. Forexample, destruction of the immune system in individuals infected withthe human immunodeficiency virus (HIV) is thought to progress throughinfected CD4⁺ T cells (about 1 in 100,000) instructing uninfected sistercells to undergo apoptosis.

Some cancers that arise by non-viral means have also developedmechanisms to escape destruction by apoptosis. Melanoma cells, forinstance, avoid apoptosis by inhibiting the expression of the geneencoding Apaf-1. Other cancer cells, especially lung and colon cancercells, secrete high levels of soluble decoy molecules that inhibit theinitiation of CTL mediated clearance of aberrant cells. Faultyregulation of the apoptotic machinery has also been implicated invarious degenerative conditions and vascular diseases.

Controlled regulation of the apoptotic process and its cellularmachinery is important to the survival of multicellular organisms.Typically, the biochemical changes that occur in a cell instructed toundergo apoptosis occur in an orderly procession. However, as shownabove, flawed regulation of apoptosis can cause serious deleteriouseffects in the organism.

The need exists for improved compositions and methods for regulating theapoptotic processes in subjects afflicted with diseases and conditionscharacterized by faulty regulation of these processes (e.g., viralinfections, hyperproliferative autoimmune disorders, chronicinflammatory conditions, and cancers). The present invention addressesthis need and provides other related advantages.

SUMMARY

The invention provides trifluoromethyl pyrazolyl guanidine compoundsthat inhibit F₁F₀-ATPase (e.g., mitochondrial F₁F₀-ATPase),pharmaceutical compositions comprising trifluoromethyl pyrazolylguanidine compounds, and methods of using such compounds andpharmaceutical compositions to treat a number of medical conditions. Theinvention relates in part to the discovery of compositions havingsurprising superior pharmaceutical properties. For example, the compound3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamide(Compound 1) was surprisingly discovered to have reduced off-targeteffects compared to3-chloro-N-(((3-chlorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamide(Compound 2). For instance, Compound 1 was surprisingly discovered to beapproximately 3-fold less potent towards Cytochrome P450 2D6 enzymecompared to Compound 2. The reduced potency of Compound 1 to CytochromeP450 2D6 enzyme improves the safety profile of the compound in patientstaking additional medication, either as part of a combination therapywith Compound 1 to treat a medical disorder described herein or for thetreatment of a different ailment. Other advantages and benefits of thecompositions and methods are described in, for example, the detaileddescription and Examples.

Accordingly, one aspect of the invention provides a family of compoundsrepresented by Formula I:

or a geometric isomer or tautomer; or a pharmaceutically acceptable saltor solvate of any of the foregoing; wherein R¹ is a chloro group locatedat either the meta-position or para-position of the phenyl group towhich it is attached.

Another aspect of the invention provides a compound represented by:

or a geometric isomer or tautomer; or a pharmaceutically acceptable saltof any of the foregoing.

Another aspect of the invention provides a compound represented by:

or a geometric isomer or tautomer; or a solvate of any of the foregoing.

The invention also provides compounds in crystalline form. For example,one aspect of the invention provides crystalline compound3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamide. Incertain embodiments, the crystalline compound3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamideis in polymorphic Form I, which exhibits an X-ray powder diffractionpattern comprising peaks at the following diffraction angles (2θ):6.2±0.2, 7.1±0.2, 9.4±0.2, 10.7±0.2, 15.6±0.2, 19.0±0.2, 20.0±0.2, and25.2±0.2. In certain other embodiments, the crystalline compound3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamideis in polymorphic Form II, which exhibits an X-ray powder diffractionpattern comprising peaks at the following diffraction angles (2θ):6.3±0.2, 7.3±0.2, 11.0±0.2, 12.8±0.2, 16.9±0.2, 19.2±0.2, 20.6±0.2,22.2±0.2, 25.7±0.2, 26.0±0.2, and 35.7±0.2.

Another aspect of the invention provides crystalline compound3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamideacetone solvate. In certain embodiments, the crystalline compound3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamideacetone solvate exhibits an X-ray powder diffraction pattern comprisingpeaks at the following diffraction angles (2θ): 6.2±0.2, 8.3±0.2,14.7±0.2, 17.5±0.2, 17.9±0.2, 18.5±0.2, 20.2±0.2, 20.9±0.2, 23.5±0.2,and 25.9±0.2.

The foregoing compounds can be present in a pharmaceutical compositioncomprising a compound described herein and a pharmaceutically acceptablecarrier.

Another aspect of the invention provides a method of treating a subjectsuffering from a medical disorder. The method comprises administering tothe subject a therapeutically effective amount of one or moretrifluoromethyl pyrazolyl guanidine compounds described herein, e.g., acompound of Formula I or one of the crystalline forms described herein,in order to ameliorate a symptom of the disorder. A large number ofdisorders can be treated using the trifluoromethyl pyrazolyl guanidinecompounds described herein. For example, the compounds described hereincan be used to treat an immune disorder or inflammatory disorder, suchas rheumatoid arthritis, psoriasis, chronic graft-versus-host disease,acute graft-versus-host disease, Crohn's disease, inflammatory boweldisease, multiple sclerosis, systemic lupus erythematosus, Celiac Sprue,idiopathic thrombocytopenic thrombotic purpura, myasthenia gravis,Sjogren's syndrome, scleroderma, ulcerative colitis, asthma, epidermalhyperplasia, and other medical disorders described herein. The compoundsdescribed herein can also be used to treat a cardiovascular disease,myeloma, lymphoma, cancer, or bacterial infection.

Another aspect of the invention provides a method of inhibiting anF₁F₀-ATPase, for example, a mitochondrial F₁F₀-ATPase. The methodcomprises exposing the F₁F₀-ATPase to a compound described herein, e.g.,a compound of Formula I or one of the crystalline forms describedherein, to inhibit said F₁F₀-ATPase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an X-ray powder diffractogram of3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamideacetone solvate, as further described in Example 2.

FIG. 2 is a differential scanning calorimetry (DSC) curve of3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamideacetone solvate, along with a thermal gravimetric analysis (TGA) curveof3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamideacetone solvate, each as further described in Example 2.

FIG. 3 is an X-ray powder diffractogram of crystalline Form I of3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamide,as further described in Example 3.

FIG. 4 is a differential scanning calorimetry curve (DSC) of crystallineForm I of3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamide,along with a thermal gravimetric analysis (TGA) curve of crystallineForm I of3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamide,each as further described in Example 3.

FIG. 5 is an X-ray powder diffractogram of crystalline Form II of3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamide,as further described in Example 4.

FIG. 6 is a differential scanning calorimetry (DSC) curve of crystallineForm II of3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamide,as further described in Example 4.

FIG. 7 is an X-ray powder diffractogram of crystalline Form II of3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamide,as further described in Example 5.

FIG. 8 is a differential scanning calorimetry curve of crystalline FormII of3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamide,as further described in Example 5.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides trifluoromethyl pyrazolyl guanidine compoundsthat inhibit F₁F₀-ATPase (e.g., mitochondrial F₁F₀-ATPase),pharmaceutical compositions comprising the trifluoromethyl pyrazolylguanidine compounds, and methods of using the trifluoromethyl pyrazolylguanidine compounds and pharmaceutical compositions in therapy. Asexplained in part above, one aspect of the invention pertains to thediscovery of compositions having surprising superior pharmaceuticalproperties. For example, the compound3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamide(Compound 1) was surprisingly discovered to have reduced off-targeteffects compared to3-chloro-N-(((3-chlorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamide(Compound 2). For instance, Compound 1 was surprisingly discovered to beapproximately 3-fold less potent towards Cytochrome P450 2D6 enzymecompared to Compound 2.

The reduced potency of Compound 1 to Cytochrome P450 2D6 enzyme improvesthe safety profile of the compound in patients taking additionalmedication, either as part of a combination therapy with Compound 1 totreat a medical disorder described herein or for the treatment of adifferent ailment. Cytochrome P450 2D6 is involved in the metabolism ofmany therapeutic agents. Inhibiting the action of Cytochrome P450 2D6 isundesirable because such inhibition can alter the rate of metabolism ofa therapeutic agent causing an otherwise safe dosing amount or frequencyto become unsafe for the patient. The present invention providescompositions with reduced affects on the Cytochrome P450 2D6 enzyme,thereby providing a therapeutic with an improved safety profile whenadministered to patients taking other medication.

Exemplary compositions and methods of the present invention aredescribed in more detail in the following sections: I. Modulators ofF₁F₀-ATPase Activity; II. Trifluoromethyl Pyrazolyl Guanidine Compounds;III. Therapeutic Applications of Trifluoromethyl Pyrazolyl GuanidineCompounds, and IV. Pharmaceutical Compositions, Formulations, andExemplary Administration Routes and Dosing Considerations. Aspects ofthe invention described in one particular section are not to be limitedto any particular section.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of organic chemistry, pharmacology,molecular biology (including recombinant techniques), cell biology,biochemistry, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature, such as“Comprehensive Organic Synthesis” (B. M. Trost & I. Fleming, eds.,1991-1992); “Molecular cloning: a laboratory manual” Second Edition(Sambrook et al., 1989); “Oligonucleotide synthesis” (M. J. Gait, ed.,1984); “Animal cell culture” (R. I. Freshney, ed., 1987); the series“Methods in enzymology” (Academic Press, Inc.); “Handbook ofexperimental immunology” (D. M. Weir & C. C. Blackwell, eds.); “Genetransfer vectors for mammalian cells” (J. M. Miller & M. P. Calos, eds.,1987); “Current protocols in molecular biology” (F. M. Ausubel et al.,eds., 1987, and periodic updates); “PCR: the polymerase chain reaction”(Mullis et al., eds., 1994); and “Current protocols in immunology” (J.E. Coligan et al., eds., 1991), each of which is herein incorporated byreference in its entirety.

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below.

As used herein, the term “guanidine” refers to a compound having thefollowing core structure:

including pharmaceutically acceptable salt forms. The symbol “

” indicates a point of attachment.

Compounds of the disclosure may contain one or more double bonds and,therefore, exist as geometric isomers. Unless indicated otherwise,generic chemical structures and graphical representations of specificcompounds encompass all geometric isomers. The present inventionencompasses the various geometric isomers and mixtures thereof resultingfrom the arrangement of substituents around a double bond (e.g., acarbon-carbon double bond, or a carbon-nitrogen double bond).Substituents around a double bond are designated as being in the “Z” or“E” configuration wherein the terms “Z” and “E” are used in accordancewith IUPAC standards. Unless otherwise specified, structures depictingdouble bonds encompass both the “E” and “Z” isomers. Substituents arounda double bond alternatively can be referred to as “cis” or “trans,”where “cis” represents substituents on the same side of the double bondand “trans” represents substituents on opposite sides of the doublebond.

Certain compounds described herein may exist as a single tautomer or asa mixture of tautomers. For example, certain guanidine compounds havinga hydrogen atom attached to at least one of the guanidine nitrogen atomscan exist as a single tautomer or a mixture of tautomers. To illustrate,depending upon the substituents attached at the R¹, R² and R³ positions,the guanidine compound may exist as a single tautomer represented by A,B, or C, or as mixture of two or more of A, B, and C.

The compounds disclosed herein can exist in solvated as well asunsolvated forms with pharmaceutically acceptable solvents such aswater, ethanol, acetone, and the like, and it is intended that theinvention embrace both solvated and unsolvated forms.

The invention also embraces isotopically labeled compounds of theinvention which are identical to those recited herein, except that oneor more atoms are replaced by an atom having an atomic mass or massnumber different from the atomic mass or mass number usually found innature. Examples of isotopes that can be incorporated into compounds ofthe invention include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorus, sulfur, fluorine and chlorine, such as ²H, ³H, ¹³C, ¹⁴C,¹⁵N, ¹⁸O, ¹⁷O ³¹P, ³²P, ³⁵S, ¹⁸F and ³⁶Cl, respectively.

Certain isotopically-labeled disclosed compounds (e.g., those labeledwith ³H and ¹⁴C) are useful in compound and/or substrate tissuedistribution assays. Tritiated (i.e., ³H) and carbon-14 (i.e., ¹⁴C)isotopes are particularly preferred for their ease of preparation anddetectability. Further, substitution with heavier isotopes such asdeuterium (i.e., ²H) may afford certain therapeutic advantages resultingfrom greater metabolic stability (e.g., increased in vivo half-life orreduced dosage requirements) and hence may be preferred in somecircumstances. Isotopically labeled compounds of the invention cangenerally be prepared by following procedures analogous to thosedisclosed in, for instance, Examples herein, by substituting anisotopically labeled reagent for a non-isotopically labeled reagent.

The chemical name“3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamide”refers to the following compound:

The terms ortho, meta and para are art-recognized and refer to 1,2-,1,3- and 1,4-disubstituted benzenes, respectively. For example, thenames 1,2-dimethylbenzene and ortho-dimethylbenzene are synonymous.

The term “IC₅₀” is art-recognized and refers to the concentration of acompound that is required for 50% inhibition of its target.

The term “EC₅₀” is art-recognized and refers to the concentration of acompound at which 50% of its maximal effect is observed.

The terms “subject” and “patient” refer to organisms to be treated bythe methods of the present invention. Such organisms preferably include,but are not limited to, mammals (e.g., murines, simians, equines,bovines, porcines, canines, felines, and the like), and most preferablyincludes humans. In the context of the invention, the terms “subject”and “patient” generally refer to an individual who will receive or whohas received treatment (e.g., administration of a compound of thepresent invention and optionally one or more other agents) for acondition characterized by the dysregulation of apoptotic processes.

As used herein, the term “effective amount” refers to the amount of acompound sufficient to effect beneficial or desired results. Aneffective amount can be administered in one or more administrations,applications or dosages and is not intended to be limited to aparticular formulation or administration route. As used herein, the term“treating” includes any effect, e.g., lessening, reducing, modulating,ameliorating or eliminating, that results in the improvement of thecondition, disease, disorder, and the like, or ameliorating a symptomthereof.

The phrase “pathologically proliferating or growing cells” refers to alocalized population of proliferating cells in an animal that is notgoverned by the usual limitations of normal growth.

As used herein, the term “un-activated target cell” refers to a cellthat is either in the G_(o) phase or one to which a stimulus has notbeen applied.

As used herein, the term “activated target lymphoid cell” refers to alymphoid cell that has been primed with an appropriate stimulus to causea signal transduction cascade, or alternatively, a lymphoid cell that isnot in G_(o) phase. Activated lymphoid cells may proliferate, undergoactivation induced cell death, or produce one or more cytotoxins,cytokines, or other related membrane-associated proteins characteristicof the cell type (e.g., CD8⁺ or CD4⁺). They are also capable ofrecognizing and binding any target cell that displays a particularantigen on its surface, and subsequently releasing its effectormolecules.

As used herein, the term “activated cancer cell” refers to a cancer cellthat has been primed with an appropriate stimulus to cause signaltransduction. An activated cancer cell may or may not be in the G_(O)phase.

An activating agent is a stimulus that upon interaction with a targetcell results in a signal transduction cascade. Examples of activatingstimuli include, but are not limited to, small molecules, radiantenergy, and molecules that bind to cell activation cell surfacereceptors. Responses induced by activation stimuli can be characterizedby changes in, among others, intracellular Ca²⁺, superoxide, or hydroxylradical levels; the activity of enzymes like kinases or phosphatases; orthe energy state of the cell. For cancer cells, activating agents alsoinclude transforming oncogenes.

As used herein, the term “dysregulation of the process of cell death”refers to any aberration in the ability (e.g., predisposition) of a cellto undergo cell death via either necrosis or apoptosis. Dysregulation ofcell death is associated with or induced by a variety of conditions,including for example, immune disorders (e.g., systemic lupuserythematosus, autoimmune disorders, rheumatoid arthritis,graft-versus-host disease, myasthenia gravis, Sjögren's syndrome, etc.),chronic inflammatory conditions (e.g., psoriasis, asthma and Crohn'sdisease), hyperproliferative disorders (e.g., tumors, B cell lymphomas,T cell lymphomas, etc.), viral infections (e.g., herpes, papilloma,HIV), and other conditions such as osteoarthritis and atherosclerosis.

It should be noted that when the dysregulation is induced by orassociated with a viral infection, the viral infection may or may not bedetectable at the time dysregulation occurs or is observed. That is,viral-induced dysregulation can occur even after the disappearance ofsymptoms of viral infection.

A “hyperproliferative disorder” as used herein refers to any conditionin which a localized population of proliferating cells in an animal isnot governed by the usual limitations of normal growth. Examples ofhyperproliferative disorders include tumors, neoplasms, lymphomas andthe like. A neoplasm is said to be benign if it does not undergoinvasion or metastasis and malignant if it does either of these. Ametastatic cell or tissue means that the cell can invade and destroyneighboring body structures. Hyperplasia is a form of cell proliferationinvolving an increase in cell number in a tissue or organ, withoutsignificant alteration in structure or function. Metaplasia is a form ofcontrolled cell growth in which one type of fully differentiated cellsubstitutes for another type of differentiated cell. Metaplasia canoccur in epithelial or connective tissue cells. A typical metaplasiainvolves a somewhat disorderly metaplastic epithelium.

The pathological growth of activated lymphoid cells often results in animmune disorder or a chronic inflammatory condition. As used herein, theterm “immune disorder” refers to any condition in which an organismproduces antibodies or immune cells which recognize the organism's ownmolecules, cells or tissues. Non-limiting examples of immune disordersinclude autoimmune disorders, immune hemolytic anemia, immune hepatitis,Berger's disease or IgA nephropathy, Celiac Sprue, chronic fatiguesyndrome, Crohn's disease, dermatomyositis, fibromyalgia,graft-versus-host disease, Grave's disease, Hashimoto's thyroiditis,idiopathic thrombocytopenia purpura, lichen planus, multiple sclerosis,myasthenia gravis, psoriasis, rheumatic fever, rheumatic arthritis,scleroderma, Sjorgren syndrome, systemic lupus erythematosus, type 1diabetes, ulcerative colitis, vitiligo, tuberculosis, and the like.

As used herein, the term “chronic inflammatory condition” refers to acondition wherein the organism's immune cells are activated. Such acondition is characterized by a persistent inflammatory response withpathologic sequelae. This state is characterized by infiltration ofmononuclear cells, proliferation of fibroblasts and small blood vessels,increased connective tissue, and tissue destruction. Examples of chronicinflammatory diseases include, but are not limited to, Crohn's disease,psoriasis, chronic obstructive pulmonary disease, inflammatory boweldisease, multiple sclerosis, and asthma. Immune diseases such asrheumatoid arthritis and systemic lupus erythematosus can also result ina chronic inflammatory state.

As used herein, the term “co-administration” refers to theadministration of at least two agent(s) (e.g., a compound of the presentinvention) or therapies to a subject. In some embodiments, theco-administration of two or more agents/therapies is concurrent. Inother embodiments, a first agent/therapy is administered prior to asecond agent/therapy. Those of skill in the art understand that theformulations and/or routes of administration of the variousagents/therapies used may vary. The appropriate dosage forco-administration can be readily determined by one skilled in the art.In some embodiments, when agents/therapies are co-administered, therespective agents/therapies are administered at lower dosages thanappropriate for their administration alone. Thus, co-administration isespecially desirable in embodiments where the co-administration of theagents/therapies lowers the requisite dosage of a known potentiallyharmful (e.g., toxic) agent(s).

As used herein, the term “pharmaceutical composition” refers to thecombination of an active agent with a carrier, inert or active, makingthe composition especially suitable for diagnostic or therapeutic use invivo or ex vivo.

As used herein, the term “pharmaceutically acceptable carrier” refers toany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, emulsions (e.g., such as an oil/wateror water/oil emulsions), and various types of wetting agents. Thecompositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants, see e.g., Martin,Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton,Pa. [1975].

As used herein, the term “pharmaceutically acceptable salt” refers toany pharmaceutically acceptable salt (e.g., acid or base) of a compoundof the present invention which, upon administration to a subject, iscapable of providing a compound of this invention or an activemetabolite or residue thereof. As is known to those of skill in the art,“salts” of the compounds of the present invention may be derived frominorganic or organic acids and bases. Examples of acids include, but arenot limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric,fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic,toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic,ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic,benzenesulfonic acid, and the like. Other acids, such as oxalic, whilenot in themselves pharmaceutically acceptable, may be employed in thepreparation of salts useful as intermediates in obtaining the compoundsof the invention and their pharmaceutically acceptable acid additionsalts.

Examples of bases include, but are not limited to, alkali metals (e.g.,sodium) hydroxides, alkaline earth metals (e.g., magnesium), hydroxides,ammonia, and compounds of formula NW₄ ⁺, wherein W is C₁₋₄ alkyl, andthe like.

Examples of salts include, but are not limited to: acetate, adipate,alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate,citrate, camphorate, camphorsulfonate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate,pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate,succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like.Other examples of salts include anions of the compounds of the presentinvention compounded with a suitable cation such as Na⁺, NH₄ ⁺, and NW₄⁺ (wherein W is a C₁₋₄ alkyl group), and the like.

For therapeutic use, salts of the compounds of the present invention arecontemplated as being pharmaceutically acceptable. However, salts ofacids and bases that are non-pharmaceutically acceptable may also finduse, for example, in the preparation or purification of apharmaceutically acceptable compound.

As used herein, the term “modulate” refers to the activity of a compound(e.g., a compound of the present invention) to affect (e.g., to promoteor retard) an aspect of cellular function, including, but not limitedto, cell growth, proliferation, apoptosis, and the like.

Throughout the description, where compositions are described as having,including, or comprising specific components, or where processes andmethods are described as having, including, or comprising specificsteps, it is contemplated that, additionally, there are compositions ofthe present invention that consist essentially of, or consist of, therecited components, and that there are processes and methods accordingto the present invention that consist essentially of, or consist of, therecited processing steps.

As a general matter, compositions specifying a percentage are by weightunless otherwise specified. Further, if a variable is not accompanied bya definition, then the previous definition of the variable controls.

I. Modulators of F₁F₀-ATPase Activity

In some embodiments, the present invention regulates F₁F₀-ATPaseactivity (e.g., mitochondrial F₁F₀-ATPase activity) through the exposureof cells to compounds of the present invention. In some embodiments, thecompounds inhibit ATP synthesis and ATP hydrolysis. The effect of thecompounds can be measured by detecting any number of cellular changes.For example, mitochondrial F₁F₀-ATPase activity and/or cell death may beassayed as described herein and in the art. In some embodiments, celllines are maintained under appropriate cell culturing conditions (e.g.,gas (CO₂), temperature and media) for an appropriate period of time toattain exponential proliferation without density dependent constraints.Cell number and or viability are measured using standard techniques,such as trypan blue exclusion/hemo-cytometry, or an Alamar Blue or MTTdye conversion assay. Alternatively, the cell may be analyzed for theexpression of genes or gene products associated with aberrations inapoptosis or necrosis.

In some embodiments, exposing the compounds of the present invention toa cell induces apoptosis. In certain other embodiments, the presentinvention induces apoptosis or arrest of cell proliferation throughinteracting with the mitochondrial F₁F₀-ATPase. In yet otherembodiments, compounds of the present invention cause an initialincrease in cellular ROS levels (e.g., O₂ ⁻) when administered to asubject.

II. Trifluoromethyl Pyrazolyl Guanidine Compounds

One aspect of the invention provides a family of compounds representedby Formula I:

or geometric isomer or tautomer; or a pharmaceutically acceptable saltor solvate of any of the foregoing; wherein R¹ is a chloro group locatedat either the meta-position or para-position of the phenyl group towhich it is attached. It is understood that the terms “meta” and “para”refer to 3-position and 4-position, respectively, of the phenyl groupwith the 1-position being the point of attachment to the carbonyl.

In certain embodiments, the compound is a compound of Formula I or ageometric isomer or tautomer, or a pharmaceutically acceptable salt ofany of the foregoing. In certain other embodiments, the compound is acompound of Formula I or a geometric isomer or tautomer, or a solvate ofany of the foregoing. In certain embodiments, the compound is in theform of an acetone solvate.

In certain embodiments, the compound is represented by:

or a geometric isomer or tautomer; or a pharmaceutically acceptable saltof any of the foregoing. In certain other embodiments, the compound isrepresented by:

or a geometric isomer or tautomer; or a solvate of any of the foregoing.In certain embodiments, the compound is in the form of an acetonesolvate.

In yet other embodiments, the compound is represented by:

or a geometric isomer or tautomer. In still other embodiments, thecompound is represented by:

Another aspect of the invention provides a compound represented by:

or a geometric isomer or tautomer; or a pharmaceutically acceptable saltor solvate of any of the foregoing. In certain embodiments, the compoundis

or a geometric isomer or tautomer; or a pharmaceutically acceptable saltof any of the foregoing.In certain certain other embodiments, the compound is

or a geometric isomer or tautomer; or a solvate of any of the foregoing.In yet other embodiments, the compound is

or a geometric isomer or tautomer. In still other embodiments, thecompound is

Crystalline Forms of Compounds

Another aspect of the invention provides compounds in crystalline form.For example, another aspect of the invention provides crystallinecompound3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamide.This crystalline compound can be in different polymeric forms. Forexample, polymorphic Form I and polymorphic Form II have beendiscovered.

A. Crystalline Form I of3-Chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamide

Procedures for making crystalline Form I of3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamideare described in, for instance, Example 3 herein. An X-ray powderdiffractogram of Form I is provided in FIG. 3. A differential scanningcalorimetry curve of Form I is provided in FIG. 4, along with a thermalgravimetric analysis curve of Form I. Form I of3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamidecan be characterized according its X-ray powder diffractogram and itsmelting point onset as determined by differential scanning calorimetry.

Accordingly, one aspect of the invention provides a crystalline3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamideexhibiting an X-ray powder diffraction pattern comprising peaks at thefollowing diffraction angles (2θ): 6.2±0.2, 7.1±0.2, 9.4±0.2, 10.7±0.2,15.6±0.2, 19.0±0.2, 20.0±0.2, and 25.2±0.2. In certain embodiments, therelative intensity of the peak at said diffraction angles (2θ) is atleast 20%. In certain embodiments, the relative intensity of the peak atsaid diffraction angles (2θ) is at least 30%.

The crystalline compound may be further characterized by the followingX-ray powder diffraction pattern expressed in terms of diffraction angle2θ, inter-planar distances d, and relative intensity (expressed as apercentage with respect to the most intense peak):

Angle [2θ] d-spacing [Å] Relative Intensity [%] 6.2 14.3 53.8 7.1 12.469.6 9.4 9.4 53.8 10.7 8.3 51.0 12.9 6.9 34.6 15.6 5.7 92.8 16.4 5.429.1 17.9 4.9 44.0 19.0 4.7 100.0 20.0 4.4 62.6 21.9 4.1 38.9 23.6 3.851.4 25.2 3.5 57.8 27.3 3.3 24.0 28.4 3.1 15.8 35.0 2.6 10.0

In still further embodiments, crystalline Form I may be furthercharacterized as having an X-ray powder diffraction patternsubstantially as shown in FIG. 3.

An X-ray powder diffraction pattern may be obtained using CuKαradiation. The temperature at which the X-ray powder diffraction patternis obtained may be, for example, 25±2 degrees Celsius.

Crystalline Form I may be further characterized by its temperature ofmelting point onset as determined by differential scanning calorimetry.In certain embodiments, the crystalline compound has a melting pointonset as determined by differential scanning calorimetry in the range offrom about 149 degrees Celsius to about 164 degrees Celsius. In certainother embodiments, the crystalline compound has a melting point onset asdetermined by differential scanning calorimetry in the range of fromabout 157 degrees Celsius to about 161 degrees Celsius. In yet otherembodiments, the crystalline compound has a melting point onset asdetermined by differential scanning calorimetry at about 159 degreesCelsius. Further yet, crystalline Form I may be further characterized byhaving a differential scanning calorimetry curve substantially the sameas shown in FIG. 4.

B. Crystalline Form II of3-Chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)aminomethylene)benzamide

Procedures for making crystalline Form II of3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamideare described in, for instance, Examples 4 and 5 herein. Exemplary X-raypowder diffractograms of crystalline Form II are provided in FIGS. 5 and7. Differential scanning calorimetry curves of crystalline Form II areprovided in FIGS. 6 and 8. Crystalline Form II of3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamidecan be characterized according its X-ray powder diffractogram and itsmelting point onset as determined by differential scanning calorimetry.

Accordingly, one aspect of the invention provides a crystalline3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamideexhibiting an X-ray powder diffraction pattern comprising peaks at thefollowing diffraction angles (2θ): 6.3±0.2, 7.3±0.2, 11.0±0.2, 12.8±0.2,16.9±0.2, 19.2±0.2, 20.6±0.2, 22.2±0.2, 25.7±0.2, 26.0±0.2, and35.7±0.2. In certain embodiments, the relative intensity of the peak atsaid diffraction angles (2θ) is at least 15%. In certain otherembodiments, the relative intensity of the peak at said diffractionangles (2θ) is at least 18%.

In certain embodiments, crystalline Form II may be further characterizedas comprising the following (expressed in terms of diffraction angle 2θ,inter-planar distances d, and relative intensity (expressed as apercentage with respect to the most intense peak)) features in an X-raypowder diffraction pattern:

Angle [2θ] d-spacing [Å] Relative Intensity [%] 6.33 13.95 65.8 7.3212.06 100.0 9.73 9.08 14.8 11.03 8.01 55.9 12.76 6.93 18.3 16.07 5.5112.7 16.91 5.24 27.2 19.18 4.62 42.0 20.56 4.31 19.8 22.20 4.00 88.422.51 3.95 43.5 23.11 3.85 56.1 23.55 3.77 29.0 25.65 3.47 48.8 25.963.43 100.0 26.75 3.33 26.2 28.03 3.18 52.6 29.03 3.07 44.7 29.51 3.0229.3 30.45 2.93 20.0 31.82 2.81 20.4 32.27 2.77 29.9 35.66 2.52 59.6

Crystalline Form II may be further characterized by the following X-raypowder diffraction pattern expressed in terms of diffraction angle 2θ,inter-planar distances d, and relative intensity (expressed as apercentage with respect to the most intense peak):

Angle [2θ] d-spacing [Å] Relative Intensity [%] 6.33 13.95 65.8 7.3212.06 100.0 9.73 9.08 14.8 11.03 8.01 55.9 12.76 6.93 18.3 13.27 6.679.7 14.75 6.00 9.3 16.07 5.51 12.7 16.91 5.24 27.2 18.45 4.80 9.9 19.184.62 42.0 19.53 4.54 8.3 20.56 4.31 19.8 22.20 4.00 88.4 22.51 3.95 43.523.11 3.85 56.1 23.55 3.77 29.0 25.65 3.47 48.8 25.96 3.43 100.0 26.753.33 26.2 28.03 3.18 52.6 29.03 3.07 44.7 29.51 3.02 29.3 30.45 2.9320.0 31.49 2.84 17.8 31.82 2.81 20.4 32.27 2.77 29.9 33.15 2.70 16.834.18 2.62 12.9 35.66 2.52 59.6 36.03 2.49 17.7 36.84 2.44 13.5 37.752.38 14.8 38.27 2.35 10.9 38.99 2.31 13.6 39.71 2.27 13.5 42.15 2.1411.2 43.46 2.08 13.7 44.19 2.05 8.3 44.55 2.03 13.1 45.96 1.97 16.346.46 1.95 16.6 48.33 1.88 9.4 49.33 1.85 9.9 50.27 1.81 9.3 50.48 1.819.9 51.25 1.78 8.2 51.45 1.77 9.5 53.86 1.70 9.1 55.63 1.65 9.8 56.541.63 6.5 57.53 1.60 8.8 59.28 1.56 6.4

In still further embodiments, crystalline Form II may be furthercharacterized as having an X-ray powder diffraction patternsubstantially as shown in FIG. 5. In other embodiments, crystalline FormII may be further characterized as having a X-ray powder diffractionpattern substantially as shown in FIG. 7.

An X-ray powder diffraction pattern may be obtained using CuKαradiation. The temperature at which the X-ray powder diffraction patternis obtained may be, for example, 25±2 degrees Celsius.

Crystalline Form II may be further characterized by its temperature ofmelting point onset as determined by differential scanning calorimetry.In certain embodiments, the crystalline compound has a melting pointonset as determined by differential scanning calorimetry in the range offrom about 158 degrees Celsius to about 165 degrees Celsius. In certainother embodiments, the crystalline compound has a melting point onset asdetermined by differential scanning calorimetry in the range of fromabout 159 degrees Celsius to about 164 degrees Celsius. In yet otherembodiments, the crystalline compound has a melting point onset asdetermined by differential scanning calorimetry at about 158 degreesCelsius, about 159 degrees Celsius, about 160 degrees Celsius, about 163degrees Celsius, about 164 degrees Celsius, or about 165 degreesCelsius. In yet other embodiments, the crystalline compound has amelting point onset as determined by differential scanning calorimetryat about 159 degrees Celsius. Further yet, crystalline Form II may befurther characterized by having a differential scanning calorimetrycurve substantially the same as shown in FIG. 6. In yet otherembodiments, crystalline Form II may be further characterized by havinga differential scanning calorimetry curve substantially the same asshown in FIG. 8.

C. Crystalline3-Chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamideAcetone Solvate

Procedures for making crystalline3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamideacetone solvate are described in, for instance, Example 2 herein. AnX-ray powder diffractogram of crystalline3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamideacetone solvate is provided in FIG. 1. A differential scanningcalorimetry curve of is provided in FIG. 2, along with a thermalgravimetric analysis curve.3-Chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamideacetone solvate can be characterized according its X-ray powderdiffractogram and its melting point onset as determined by differentialscanning calorimetry.

Accordingly, one aspect of the invention provides a crystalline3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamideacetone solvate exhibiting an X-ray powder diffraction patterncomprising peaks at the following diffraction angles (2θ): 6.2±0.2,8.3±0.2, 14.7±0.2, 17.5±0.2, 17.9±0.2, 18.5±0.2, 20.2±0.2, 20.9±0.2,23.5±0.2, and 25.9±0.2. In certain embodiments, wherein the relativeintensity in said diffraction angles (2θ) is at least 10%. In certainother embodiments, wherein the relative intensity in said diffractionangles (2θ) is at least 15%.

The crystalline compound may be further characterized by the followingX-ray powder diffraction pattern expressed in terms of diffraction angle2θ, inter-planar distances d, and relative intensity (expressed as apercentage with respect to the most intense peak):

Angle [2θ] d-spacing [Å] Relative Intensity [%] 6.2 14.3 100 7.2 12.314.4 8.3 10.7 54.6 14.7 6.0 32.6 15.7 5.6 12.6 15.8 5.6 16.2 16.8 5.313.5 17.5 5.1 39.7 17.9 5.0 20.7 18.5 4.8 23.3 19.0 4.7 23.0 19.6 4.511.4 20.1 4.4 18.1 20.2 4.4 23.5 20.4 4.3 10.8 20.9 4.3 31.0 21.9 4.111.9 23.5 3.8 20.2 23.7 3.7 13.4 24.1 3.7 12.2 24.4 3.7 21.0 24.6 3.616.0 25.3 3.5 13.4 25.9 3.4 60.9 26.5 3.4 12.9 27.9 3.2 10.9 28.1 3.211.9 29.1 3.1 14.0

In still further embodiments, crystalline3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamideacetone solvate may be further characterized as having a X-ray powderdiffraction pattern substantially as shown in FIG. 1.

An X-ray powder diffraction pattern may be obtained using CuKαradiation. The temperature at which the X-ray powder diffraction patternis obtained may be, for example, 25±2 degrees Celsius.

Crystalline3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamideacetone solvate may be further characterized by having a melting pointonset at about 159 degrees Celsius. Further yet, the crystallinecompound may be further characterized by a differential scanningcalorimetry curve substantially the same as shown in FIG. 2.

In certain other embodiments, the compound is one of the compoundslisted in Examples 1-7, or a pharmaceutically acceptable salt of saidcompounds. In certain other embodiments, the compound is one of thecompounds listed in Examples 1-8, or a pharmaceutically acceptable saltof said compounds. It is understood that the foregoing compounds can becombined with a pharmaceutically acceptable carrier to produce apharmaceutical composition.

Exemplary methods for preparing compounds described herein are providedin the examples.

III. Therapeutic Applications of Trifluoromethyl Pyrazolyl GuanidineCompounds

It is contemplated that the guanidine compounds described herein, suchas the guanidine compounds of Formula I and specific crystallinecompounds described herein, provide therapeutic benefits to patientssuffering from any one or more of a number of conditions, e.g., diseasescharacterized by dysregulation of F₁F₀-ATPase activity, diseasescharacterized by dysregulation of necrosis and/or apoptosis processes ina cell or tissue, and diseases characterized by aberrant cell growthand/or hyperproliferation. The compounds described herein can also beused to treat a variety of dysregulatory disorders related to cellulardeath as described elsewhere herein. Additionally, the compoundsdescribed herein can be used to inhibit ATP synthesis.

Accordingly, one aspect of the invention provides a method of treating asubject suffering from a medical disorder. The method comprisesadministering to the subject a therapeutically effective amount of oneor more guanidine compounds described herein, e.g., a compound ofFormula I or specific crystalline compound, as described in Section IIabove, in order to ameliorate a symptom of the disorder.

A large number of medical disorders can be treated using the guanidinecompounds described herein. For example, the compounds described hereincan be used to treat medical disorders characterized by dysregulation ofnecrosis and/or apoptosis processes in a cell or tissue, diseasescharacterized by aberrant cell growth and/or hyperproliferation, etc.,or lupus, rheumatoid arthritis, psoriasis, graft-versus-host disease,Crohn's disease, inflammatory bowel disease, multiple sclerosis,cardiovascular disease, myeloma, lymphoma, cancer, and bacterialinfection. In certain embodiments, the cancer is a solid tumor,leukemia, colon cancer, pancreatic cancer, breast cancer, ovariancancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, lungcancer, small cell lung cancer, non-small cell lung cancer, bladdercancer, stomach cancer, cervical cancer, testicular tumor, skin cancer,rectal cancer, thyroid cancer, kidney cancer, uterus cancer, espophaguscancer, liver cancer, an acoustic neuroma, oligodendroglioma,meningioma, melanoma, neuroblastoma, or retinoblastoma.

Although not wishing to be bound to a particular theory, it is believedthat the compounds impart therapeutic benefit by modulating (e.g.,inhibiting or promoting) the activity of the F₁F₀-ATPase complexes(e.g., mitochondrial F₁F₀-ATPase complexes) in affected cells ortissues. In some embodiments, the compositions of the present inventionare used to treat immune/chronic inflammatory conditions (e.g.,psoriasis, autoimmune disorders, organ-transplant rejection, andepidermal hyperplasia). In further embodiments, the compositions of thepresent invention are used in conjunction with stenosis therapy to treatcompromised (e.g., occluded) vessels.

In certain embodiments, a composition comprising a guanidine compound isadministered under conditions (e.g., timing, dose, co-administrationwith other agent, mode of administration, selection of subject, use oftargeting agents, etc.) that maximize desired effects directed at theF₁F₀-ATPase.

In certain embodiments, the medical disorder is an immune disorder. Incertain other embodiments, the medical disorder is an inflammatorydisorder. In certain other embodiments, the medical disorder is anautoimmune disorder. In certain other embodiments, the medical disorderis rheumatoid arthritis, psoriasis, chronic graft-versus-host disease,acute graft-versus-host disease, Crohn's disease, inflammatory boweldisease, multiple sclerosis, systemic lupus erythematosus, Celiac Sprue,idiopathic thrombocytopenic thrombotic purpura, myasthenia gravis,Sjogren's syndrome, scleroderma, ulcerative colitis, asthma, uveitis, orepidermal hyperplasia.

In certain embodiments, the medical disorder is Crohn's disease orulcerative colitis.

In certain other embodiments, the medical disorder is cartilageinflammation, bone degradation, arthritis, juvenile arthritis, juvenilerheumatoid arthritis, pauciarticular juvenile rheumatoid arthritis,polyarticular juvenile rheumatoid arthritis, systemic onset juvenilerheumatoid arthritis, juvenile ankylosing spondylitis, juvenileenteropathic arthritis, juvenile reactive arthritis, juvenile Reter'sSyndrome, SEA Syndrome, juvenile dermatomyositis, juvenile psoriaticarthritis, juvenile scleroderma, juvenile systemic lupus erythematosus,juvenile vasculitis, pauciarticular rheumatoid arthritis, polyarticularrheumatoid arthritis, systemic onset rheumatoid arthritis, ankylosingspondylitis, enteropathic arthritis, reactive arthritis, Reter'sSyndrome, dermatomyositis, psoriatic arthritis, vasculitis, myolitis,polymyolitis, dermatomyolitis, osteoarthritis, polyarteritis nodossa,Wegener's granulomatosis, arteritis, polymyalgia rheumatica,sarcoidosis, sclerosis, primary biliary sclerosis, sclerosingcholangitis, dermatitis, atopic dermatitis, atherosclerosis, Still'sdisease, chronic obstructive pulmonary disease, Guillain-Barre disease,Type I diabetes mellitus, Graves' disease, Addison's disease, Raynaud'sphenomenon, or autoimmune hepatitis. In certain embodiments, thepsoriasis is plaque psoriasis, guttate psoriasis, inverse psoriasis,pustular psoriasis, or erythrodermic psoriasis.

In certain other embodiments, the medical disorder is Crohn's disease,inflammatory bowel disease, multiple sclerosis, graft-versus-hostdisease, lupus, rheumatoid arthritis, or psoriasis. In certain otherembodiments, the medical disorder is cardiovascular disease, myeloma,lymphoma, or cancer. In certain other embodiments, the medical disorderis lupus, rheumatoid arthritis, psoriasis, graft-versus-host disease,myeloma, or lymphoma. In certain other embodiments, the medical disorderis cardiovascular disease or cancer. In certain other embodiments, themedical disorder is Crohn's disease, inflammatory bowel disease, ormultiple sclerosis. In certain other embodiments, the medical disorderis graft-versus-host disease. In further embodiments, the medicaldisorder is a bacterial infection. In certain embodiments, the patient(or subject) is a human.

As indicated above, the guanidine compounds described herein can be usedin the treatment of a bacterial infection. A variety of bacteria arecontemplated to be susceptible to the guanidine compounds.Representative bacteria include Staphylococci species, e.g., S. aureus;Enterococci species, e.g., E. faecalis and E. faecium; Streptococcispecies, e.g., S. pyogenes and S. pneumoniae; Escherichia species, e.g.,E. coli, including enterotoxigenic, enteropathogenic, enteroinvasive,enterohemorrhagic and enteroaggregative E. coli strains; Haemophilusspecies, e.g., H. influenza; and Moraxella species, e.g., M.catarrhalis. Other examples include Mycobacteria species, e.g., M.tuberculosis, M. avian-intracellulare, M. kansasii, M bovis, M.africanum, M. genavense, M. leprae, M. xenopi, M simiae, M.scrofulaceum, M. malmoense, M celatum, M abscessus, M. chelonae, M.szulgai, M. gordonae, M. haemophilum, M. fortuni and M. marinum;Corynebacteria species, e.g., C. diphtheriae; Vibrio species, e.g., V.cholerae; Campylobacter species, e.g., C. jejuni; Helicobacter species,e.g., H. pylori; Pseudomonas species, e.g., P. aeruginosa; Legionellaspecies, e.g., L. pneumophila; Treponema species, e.g., T. pallidum;Borrelia species, e.g., B. burgdorferi; Listeria species, e.g., Lmonocytogenes; Bacillus species, e.g., B. cereus; Bordatella species,e.g., B. pertussis; Clostridium species, e.g., C. perfringens, C.tetani, C. difficile and C. botulinum; Neisseria species, e.g., N.meningitidis and N. gonorrhoeae; Chlamydia species, e.g., C. psittaci,C. pneumoniae and C. trachomatis; Rickettsia species, e.g., R.rickettsii and R. prowazekii; Shigella species, e.g., S. sonnei;Salmonella species, e.g., S. typhimurium; Yersinia species, e.g., Y.enterocolitica and Y. pseudotuberculosis; Klebsiella species, e.g., K.pneumoniae; Mycoplasma species, e.g., M. pneumoniae; and Trypanosomabrucei. In certain embodiments, the guanidine compounds described hereinare used to treat a subject suffering from a bacterial infectionselected from the group consisting of S. aureus, E. faecalis, E.faecium, S. pyogenes, S. pneumonia, and P. aeruginosa. In certainembodiments, the guanidine compounds described herein are used to treata subject suffering from a Trypanosoma brucei infection.

The antibacterial activity of the compounds described herein may beevaluated using standard assays known in the art, such as the microbrothdilution minimum inhibition concentration (MIC) assay, as furtherdescribed in National Committee for Clinical Laboratory Standards.Performance Standards for Antimicrobial Susceptibility Testing;Fourteenth Informational Supplement. NCCLS document M100-S14 {ISBN1-56238-516-X}. This assay may be used to determine the minimumconcentration of a compound necessary to prevent visible bacterialgrowth in a solution. In general, the drug to be tested is seriallydiluted into wells, and aliquots of liquid bacterial culture are added.This mixture is incubated under appropriate conditions, and then testedfor growth of the bacteria. Compounds with low or no antibiotic activity(a high MIC) will allow growth at high concentrations of compound, whilecompounds with high antibiotic activity will allow bacterial growth onlyat lower concentrations (a low MIC).

The assay uses stock bacterial culture conditions appropriate for thechosen strain of bacteria. Stock cultures from the permanent stockculture collection can be stored as frozen suspensions at −70° C.Cultures may be suspended in 10% skim milk (BD) prior to snap freezingin dry ice/ethanol and then placed in a −70° C. freezer. Cultures may bemaintained on Tryptic Soy Agar containing 5% Sheep Blood at roomtemperature (20° C.), and each culture may be recovered from frozen formand transferred an additional time before MIC testing. Fresh plates areinoculated the day before testing, incubated overnight, and checked toconfirm purity and identity.

The identity and purity of the cultures recovered from the stock culturecan be confirmed to rule out the possibility of contamination. Theidentity of the strains may be confirmed by standard microbiologicalmethods (See, e.g., Murray et al., Manual of Clinical Microbiology,Eighth Edition. ASM Press {ISBN 1-55581-255-4}). In general, culturesare streaked onto appropriate agar plates for visualization of purity,expected colony morphology, and hemolytic patterns. Gram stains can alsobe utilized. The identities are confirmed using a MicroScan WalkAway 40SI Instrument (Dade Behring, West Sacramento, Calif.). This deviceutilizes an automated incubator, reader, and computer to assess foridentification purposes the biochemical reactions carried out by eachorganism. The MicroScan WalkAway can also be used to determine apreliminary MIC, which may be confirmed using the method describedbelow.

Frozen stock cultures may be used as the initial source of organisms forperforming microbroth dilution minimum inhibition concentration (MIC)testing. Stock cultures are passed on their standard growth medium forat least 1 growth cycle (18-24 hours) prior to their use. Most bacteriamay be prepared directly from agar plates in 10 mL aliquots of theappropriate broth medium. Bacterial cultures are adjusted to the opacityof a 0.5 McFarland Standard (optical density value of 0.28-0.33 on aPerkin-Elmer Lambda EZ150 Spectrophotometer, Wellesley, Mass., set at awavelength of 600 nm). The adjusted cultures are then diluted 400 fold(0.25 mL inoculum+100 mL broth) in growth media to produce a startingsuspension of approximately 5×105 colony forming units (CFU)/mL. Mostbacterial strains may be tested in cation adjusted Mueller Hinton Broth(CAMHB).

Test compounds (“drugs”) are solubilized in a solvent suitable for theassay, such as DMSO. Drug stock solutions may be prepared on the day oftesting. Microbroth dilution stock plates may be prepared in twodilution series, 64 to 0.06 μg drug/mL and 0.25 to 0.00025 μg drug/mL.For the high concentration series, 200 μL of stock solution (2 mg/mL) isadded to duplicate rows of a 96-well microtiter plate. This is used asthe first well in the dilution series. Serial two-fold decrementaldilutions are made using a BioMek FX robot (Beckman Coulter Inc.,Fullerton, Calif.) with 10 of the remaining 11 wells, each of which willcontain 100 μL of the appropriate solvent/diluent. Row 12 containssolvent/diluent only and serves as the control. For the first well ofthe low concentration series, 200 μL of an 8 μg/mL stock are added toduplicate rows of a 96-well plate. Serial two-fold dilutions are made asdescribed above.

Daughter 96-well plates may be spotted (3.2 μL/well) from the stockplates listed above using the BioMek FX robot and used immediately orfrozen at −70° C. until use. Aerobic organisms are inoculated (100 μLvolumes) into the thawed plates using the BioMek FX robot. Theinoculated plates are be placed in stacks and covered with an emptyplate. These plates are then incubated for 16 to 24 hours in ambientatmosphere according to CLSI guidelines (National Committee for ClinicalLaboratory Standards, Methods for Dilution, Antimicrobial Tests forBacteria that Grow Aerobically; Approved Standard-Sixth Edition. NCCLSdocument M7-A6 {ISBN 1-56238-486-4}).

After inoculation and incubation, the degree of bacterial growth can beestimated visually with the aid of a Test Reading Mirror (DynexTechnologies 220 16) in a darkened room with a single light shiningdirectly through the top of the microbroth tray. The MIC is the lowestconcentration of drug that prevents macroscopically visible growth underthe conditions of the test.

Additionally, any one or more of the pyrazolyl guanidine compoundsdescribed herein can be used to treat a F₁F₀-ATP hydrolase associateddisorder (e.g., myocardial infarction, ventricular hypertrophy, coronaryartery disease, non-Q wave MI, congestive heart failure, cardiacarrhythmias, unstable angina, chronic stable angina, Prinzmetal'sangina, high blood pressure, intermittent claudication, peripheralocclusive arterial disease, thrombotic or thromboembolic symptoms ofthromboembolic stroke, venous thrombosis, arterial thrombosis, cerebralthrombosis, pulmonary embolism, cerebral embolism, thrombophilia,disseminated intravascular coagulation, restenosis, atrial fibrillation,ventricular enlargement, atherosclerotic vascular disease,atherosclerotic plaque rupture, atherosclerotic plaque formation,transplant atherosclerosis, vascular remodeling atherosclerosis, cancer,surgery, inflammation, systematic infection, artificial surfaces,interventional cardiology, immobility, medication, pregnancy and fetalloss, and diabetic complications comprising retinopathy, nephropathy andneuropathy) in a subject.

Combination Therapy

Additionally, the guanidine compounds described herein can be used incombination with at least one other therapeutic agent, such as Bz-423 (abenzodiazepine compound as described in U.S. Pat. Nos. 7,144,880 and7,125,866, U.S. patent application Ser. Nos. 11/586,097, 11/585,492,11/445,010, 11/324,419, 11/176,719, 11/110,228, 10/935,333, 10/886,450,10/795,535, 10/634,114, 10/427,211, 10/217,878, and 09/767,283, and U.S.Provisional Patent Nos. 60/878,519, 60/812,270, 60/802,394, 60/732,045,60/730,711, 60/704,102, 60/686,348, 60/641,040, 60/607,599, and60/565,788), potassium channel openers, calcium channel blockers, sodiumhydrogen exchanger inhibitors, antiarrhythmic agents,antiatherosclerotic agents, anticoagulants, antithrombotic agents,prothrombolytic agents, fibrinogen antagonists, diuretics,antihypertensive agents, ATPase inhibitors, mineralocorticoid receptorantagonists, phospodiesterase inhibitors, antidiabetic agents,anti-inflammatory agents, antioxidants, angiogenesis modulators,antiosteoporosis agents, hormone replacement therapies, hormone receptormodulators, oral contraceptives, antiobesity agents, antidepressants,antianxiety agents, antipsychotic agents, antiproliferative agents,antitumor agents, antiulcer and gastroesophageal reflux disease agents,growth hormone agents and/or growth hormone secretagogues, thyroidmimetics, anti-infective agents, antiviral agents, antibacterial agents,antifungal agents, cholesterol/lipid lowering agents and lipid profiletherapies, and agents that mimic ischemic preconditioning and/ormyocardial stunning, antiatherosclerotic agents, anticoagulants,antithrombotic agents, antihypertensive agents, antidiabetic agents, andantihypertensive agents selected from ACE inhibitors, AT-1 receptorantagonists, ET receptor antagonists, dual ET/AII receptor antagonists,vasopepsidase inhibitors, an antiplatelet agent selected from GPIIb/IIIablockers, P2Y₁ and P2Y₁₂ antagonists, thromboxane receptor antagonists,or aspirin, along with a pharmaceutically-acceptable carrier or diluentin a pharmaceutical composition.

IV. Pharmaceutical Compositions, Formulations, and ExemplaryAdministration Routes and Dosing Considerations

Exemplary embodiments of various contemplated medicaments andpharmaceutical compositions are provided below.

A. Preparing Medicaments

Compounds of the present invention are useful in the preparation ofmedicaments to treat a variety of conditions, such as conditionsassociated with dysregulation of cell death, aberrant cell growth andhyperproliferation. One of skill in the art will appreciate that any oneor more of the compounds described herein, including the many specificembodiments, are prepared by applying standard pharmaceuticalmanufacturing procedures. Such medicaments can be delivered to thesubject by using delivery methods that are well-known in thepharmaceutical arts.

B. Exemplary Pharmaceutical Compositions and Formulation

In some embodiments of the present invention, the compositions areadministered alone, while in some other embodiments, the compositionsare preferably present in a pharmaceutical formulation comprising atleast one active ingredient/agent, as discussed above, together with asolid support or alternatively, together with one or morepharmaceutically acceptable carriers and optionally other therapeuticagents (e.g., those described in section III hereinabove). Each carriershould be “acceptable” in the sense that it is compatible with the otheringredients of the formulation and not injurious to the subject.

Contemplated formulations include those suitable for oral, rectal,nasal, topical (including transdermal, buccal and sublingual), vaginal,parenteral (including subcutaneous, intramuscular, intravenous andintradermal) and pulmonary administration. In some embodiments,formulations are conveniently presented in unit dosage form and areprepared by any method known in the art of pharmacy. Such methodsinclude the step of bringing into association the active ingredient withthe carrier which constitutes one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association (e.g., mixing) the active ingredient withliquid carriers or finely divided solid carriers or both, and then ifnecessary shaping the product.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tablets,wherein each preferably contains a predetermined amount of the activeingredient; as a powder or granules; as a solution or suspension in anaqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion ora water-in-oil liquid emulsion. In other embodiments, the activeingredient is presented as a bolus, electuary, or paste, etc.

In some embodiments, tablets comprise at least one active ingredient andoptionally one or more accessory agents/carriers are made by compressingor molding the respective agents. In some embodiments, compressedtablets are prepared by compressing in a suitable machine the activeingredient in a free-flowing form such as a powder or granules,optionally mixed with a binder (e.g., povidone, gelatin,hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative,disintegrant (e.g., sodium starch glycolate, cross-linked povidone,cross-linked sodium carboxymethyl cellulose) surface-active ordispersing agent. Molded tablets are made by molding in a suitablemachine a mixture of the powdered compound (e.g., active ingredient)moistened with an inert liquid diluent. Tablets may optionally be coatedor scored and may be formulated so as to provide slow or controlledrelease of the active ingredient therein using, for example,hydroxypropylmethyl cellulose in varying proportions to provide thedesired release profile. Tablets and capsules may optionally be providedwith an enteric coating, to provide release in parts of the gut otherthan the stomach.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Pharmaceutical compositions for topical administration according to thepresent invention are optionally formulated as ointments, creams,suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosolsor oils. In alternative embodiments, topical formulations comprisepatches or dressings such as a bandage or adhesive plasters impregnatedwith active ingredient(s), and optionally one or more excipients ordiluents. In some embodiments, the topical formulations include acompound(s) that enhances absorption or penetration of the activeagent(s) through the skin or other affected areas. Examples of suchdermal penetration enhancers include dimethylsulfoxide (DMSO) andrelated analogues.

If desired, the aqueous phase of a cream base includes, for example, atleast about 30% w/w of a polyhydric alcohol, i.e., an alcohol having twoor more hydroxyl groups such as propylene glycol, butane-1,3-diol,mannitol, sorbitol, glycerol and polyethylene glycol and mixturesthereof.

In some embodiments, oily phase emulsions of this invention areconstituted from known ingredients in a known manner. This phasetypically comprises a lone emulsifier (otherwise known as an emulgent),it is also desirable in some embodiments for this phase to furthercomprise a mixture of at least one emulsifier with a fat or an oil orwith both a fat and an oil.

Preferably, a hydrophilic emulsifier is included together with alipophilic emulsifier so as to act as a stabilizer. In some embodimentsit is also preferable to include both an oil and a fat. Together, theemulsifier(s) with or without stabilizer(s) make up the so-calledemulsifying wax, and the wax together with the oil and/or fat make upthe so-called emulsifying ointment base which forms the oily dispersedphase of the cream formulations.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredient is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the agent.

Formulations for rectal administration may be presented as a suppositorywith suitable base comprising, for example, cocoa butter or asalicylate.

Formulations suitable for vaginal administration may be presented aspessaries, creams, gels, pastes, foams or spray formulations containingin addition to the agent, such carriers as are known in the art to beappropriate.

Formulations suitable for nasal administration, wherein the carrier is asolid, include coarse powders having a particle size, for example, inthe range of about 20 to about 500 microns which are administered in themanner in which snuff is taken, i.e., by rapid inhalation (e.g., forced)through the nasal passage from a container of the powder held close upto the nose. Other suitable formulations wherein the carrier is a liquidfor administration include, but are not limited to, nasal sprays, drops,or aerosols by nebulizer, and include aqueous or oily solutions of theagents.

Formulations suitable for parenteral administration include aqueous andnon-aqueous isotonic sterile injection solutions which may containantioxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents, and liposomes or other microparticulatesystems which are designed to target the compound to blood components orone or more organs. In some embodiments, the formulations arepresented/formulated in unit-dose or multi-dose sealed containers, forexample, ampoules and vials, and may be stored in a freeze-dried(lyophilized) condition requiring only the addition of the sterileliquid carrier, for example water for injections, immediately prior touse. Extemporaneous injection solutions and suspensions may be preparedfrom sterile powders, granules and tablets of the kind previouslydescribed.

Preferred unit dosage formulations are those containing a daily dose orunit, daily subdose, as herein above-recited, or an appropriate fractionthereof, of an agent.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example, those suitable for oral administration mayinclude such further agents as sweeteners, thickeners and flavoringagents. It also is intended that the agents, compositions and methods ofthis invention be combined with other suitable compositions andtherapies. Still other formulations optionally include food additives(suitable sweeteners, flavorings, colorings, etc.), phytonutrients(e.g., flax seed oil), minerals (e.g., Ca, Fe, K, etc.), vitamins, andother acceptable compositions (e.g., conjugated linoelic acid),extenders, and stabilizers, etc.

C. Exemplary Administration Routes and Dosing Considerations

Various delivery systems are known and can be used to administertherapeutic agents (e.g., exemplary compounds as described above) of thepresent invention, e.g., encapsulation in liposomes, microparticles,microcapsules, receptor-mediated endocytosis, and the like. Methods ofdelivery include, but are not limited to, intra-arterial,intra-muscular, intravenous, intranasal, and oral routes. In specificembodiments, it may be desirable to administer the pharmaceuticalcompositions of the invention locally to the area in need of treatment;this may be achieved by, for example, and not by way of limitation,local infusion during surgery, injection, or by means of a catheter.

The agents identified can be administered to subjects or individualssusceptible to or at risk of developing pathological growth of targetcells and correlated conditions. When the agent is administered to asubject such as a mouse, a rat or a human patient, the agent can beadded to a pharmaceutically acceptable carrier and systemically ortopically administered to the subject. To identify patients that can bebeneficially treated, a tissue sample is removed from the patient andthe cells are assayed for sensitivity to the agent.

Therapeutic amounts are empirically determined and vary with thepathology being treated, the subject being treated and the efficacy andtoxicity of the agent. When delivered to an animal, the method is usefulto further confirm efficacy of the agent. One example of an animal modelis MLR/MpJ-lpr/lpr (“MLR-lpr”) (available from Jackson Laboratories, BarHarbor, Me.). MLR-lpr mice develop systemic autoimmune disease.Alternatively, other animal models can be developed by inducing tumorgrowth, for example, by subcutaneously inoculating nude mice with about10⁵ to about 10⁹ hyperproliferative, cancer or target cells as definedherein. When the tumor is established, the compounds described hereinare administered, for example, by subcutaneous injection around thetumor. Tumor measurements to determine reduction of tumor size are madein two dimensions using venier calipers twice a week. Other animalmodels may also be employed as appropriate. Such animal models for theabove-described diseases and conditions are well-known in the art.

In some embodiments, in vivo administration is effected in one dose,continuously or intermittently throughout the course of treatment.Methods of determining the most effective means and dosage ofadministration are well known to those of skill in the art and vary withthe composition used for therapy, the purpose of the therapy, the targetcell being treated, and the subject being treated. Single or multipleadministrations are carried out with the dose level and pattern beingselected by the treating physician.

Suitable dosage formulations and methods of administering the agents arereadily determined by those of skill in the art. Preferably, thecompounds are administered at about 0.01 mg/kg to about 200 mg/kg, morepreferably at about 0.1 mg/kg to about 100 mg/kg, even more preferablyat about 0.5 mg/kg to about 50 mg/kg. When the compounds describedherein are co-administered with another agent (e.g., as sensitizingagents), the effective amount may be less than when the agent is usedalone.

The pharmaceutical compositions can be administered orally,intranasally, parenterally or by inhalation therapy, and may take theform of tablets, lozenges, granules, capsules, pills, ampoules,suppositories or aerosol form. They may also take the form ofsuspensions, solutions and emulsions of the active ingredient in aqueousor non-aqueous diluents, syrups, granulates or powders. In addition toan agent of the present invention, the pharmaceutical compositions canalso contain other pharmaceutically active compounds or a plurality ofcompounds of the invention.

More particularly, an agent of the present invention also referred toherein as the active ingredient, may be administered for therapy by anysuitable route including, but not limited to, oral, rectal, nasal,topical (including, but not limited to, transdermal, aerosol, buccal andsublingual), vaginal, parental (including, but not limited to,subcutaneous, intramuscular, intravenous and intradermal) and pulmonary.It is also appreciated that the preferred route varies with thecondition and age of the recipient, and the disease being treated.

Ideally, the agent should be administered to achieve peak concentrationsof the active compound at sites of disease. This may be achieved, forexample, by the intravenous injection of the agent, optionally insaline, or by oral administration, for example, as a tablet, capsule orsyrup containing the active ingredient.

Desirable blood levels of the agent may be maintained by a continuousinfusion to provide a therapeutic amount of the active ingredient withindisease tissue. The use of operative combinations is contemplated toprovide therapeutic combinations requiring a lower total dosage of eachcomponent than may be required when each individual therapeutic compoundor drug is used alone, thereby reducing adverse effects.

D. Exemplary Co-Administration Routes and Dosing Considerations

The invention also includes methods involving co-administration of thecompounds described herein with one or more additional active agents.Indeed, it is a further aspect of this invention to provide methods forenhancing prior art therapies and/or pharmaceutical compositions byco-administering a compound of this invention. In co-administrationprocedures, the agents may be administered concurrently or sequentially.In one embodiment, the compounds described herein are administered priorto the other active agent(s). The pharmaceutical formulations and modesof administration may be any of those described above. In addition, thetwo or more co-administered chemical agents, biological agents orradiation may each be administered using different modes or differentformulations.

The agent or agents to be co-administered depend on the type ofcondition being treated. For example, when the condition being treatedis cancer, the additional agent can be a chemotherapeutic agent orradiation. When the condition being treated is an immune disorder, theadditional agent can be an immunosuppressant or an anti-inflammatoryagent. When the condition being treated is chronic inflammation, theadditional agent can be an anti-inflammatory agent. The additionalagents to be co-administered, such as anticancer, immunosuppressant,anti-inflammatory, can be any of the well-known agents in the art,including, but not limited to, those that are currently in clinical use.The determination of appropriate type and dosage of radiation treatmentis also within the skill in the art or can be determined with relativeease.

Treatment of the various conditions associated with abnormal apoptosisis generally limited by the following two major factors: (1) thedevelopment of drug resistance and (2) the toxicity of known therapeuticagents. In certain cancers, for example, resistance to chemicals andradiation therapy has been shown to be associated with inhibition ofapoptosis. Some therapeutic agents have deleterious side effects,including non-specific lymphotoxicity, renal and bone marrow toxicity.

The methods described herein address both these problems. Drugresistance, where increasing dosages are required to achieve therapeuticbenefit, is overcome by co-administering the compounds described hereinwith the known agent. The compounds described herein sensitize targetcells to known agents (and vice versa) and, accordingly, less of theseagents are needed to achieve a therapeutic benefit.

The sensitizing function of the claimed compounds also addresses theproblems associated with toxic effects of known therapeutics. Ininstances where the known agent is toxic, it is desirable to limit thedosages administered in all cases, and particularly in those cases wheredrug resistance has increased the requisite dosage. When the claimedcompounds are co-administered with the known agent, they reduce thedosage required which, in turn, reduces the deleterious effects.

EXAMPLES

The invention now being generally described, will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.Unless indicated otherwise, the HPLC method used is as follows: WatersSymmetry C-18 column, 4.6×150 mm, 3.5 micron, 25° C., 1.0 mL/min, 25minute gradient of 5% MeCN in H₂O (0.1% TFA) to 95% MeCN in H₂O (0.1%TFA), then 95% MeCN in H₂O (0.1% TFA) for 10 minutes, and thenequilibration to 5% MeCN in H₂O (0.1% TFA) over 5.0 minutes. The phrase“H₂O (0.1% TFA)” is art-recognized and refers to water containing 0.1%v/v trifluoroacetic acid.

Example 1 Preparation of3-Chloro-N-((3-Chloro-5-Fluorophenyl)Carbamothioyl)Benzamide

3-Chlorobenzoyl chloride (200 g, 1.14 mol) was dissolved in acetonitrile(4.5 L) and the reaction vessel containing this mixture was cooled in anice/water bath. Potassium thiocyanate (122 g, 1.26 mol, 1.10 equiv) wasadded to the reaction mixture. The cooling bath was removed from thereaction vessel after 15 minutes and the resulting slurry was stirred atroom temperature for approximately 1 hour. Next,3-chloro-5-fluoroaniline (138 mL, 1.37 mol, 1.2 equiv) was added as asolution in acetonitrile (138 mL) over 5 minutes. The reaction mixturewas then stirred at room temperature overnight and subsequently quenchedwith water (4.30 L), and then stirred at room temperature for 1 hour.The resulting solid was collected by filtration and rinsed withacetonitrile/water (1/1 mixture by volume, 780 mL). The solid was driedat 60° C. in vacuo to provide 390 g (99% yield) of the title compound asa solid. ¹H NMR (DMSO-d₆) δ 12.5 (s, 1H), 11.9 (s, 1H), 8.01 (t, 1H,J=1.8), 7.89 (d, 1H, J=7.8), 7.69 (m, 3H), 7.56 (t, 1H, J=7.8), 7.34(dt, 1H, J=8.7, 1.8). HPLC: >99% purity at 23.73 min.

Example 2 Preparation of Crystalline3-Chloro-N-(((3-Chloro-5-Fluorophenyl)Amino)((5-(Trifluoromethyl)-1H-Pyrazol-3-Yl)Amino)Methylene)BenzamideAcetone Solvate

A suspension of3-chloro-N-((3-chloro-5-fluorophenyl)carbamothioyl)benzamide (75 g, 219mmol) in tert-butyl methyl ether (TBME) (1.1 L) was heated to 50° C.Next, 5-(trifluoromethyl)-1H-pyrazol-3-amine (39.6 g, 262 mmol, 1.20equiv) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) (64.2 g,328 mmol, 1.50 equiv) were added. The resulting reaction mixture wasstirred at 50° C. for 1 hour and diluted with EtOAc (1.0 L) and water(1.0 L). The organic extract was washed with a saturated, aqueous sodiumchloride solution (0.5 L), dried (Na₂SO₄), filtered, and concentrated.The resulting residue was diluted with EtOAc (300 mL) and this mixturewas warmed to 50° C., then filtered through Celite. Next, the filtratewas concentrated to approximately 75 mL and slowly diluted with hexanes(750 mL). The resulting mixture was stirred at room temperature for 1hour and filtered. The resulting solid was heated in acetone (100 mL),filtered, diluted with hexanes (100 mL), and the resulting mixture washeated until a clear solution developed. Next, the mixture was furtherdiluted with hexanes (300 mL) and stirred at room temperature overnight.The resulting solids were collected by filtration, rinsed with hexanes(100 mL) and dried in vacuo at room temperature to afford (25.1 g, 25%yield) of crystalline3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamideacetone solvate. ¹H NMR analysis indicates that the molar ratio of3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamideto acetone is 3 to 2. ¹H NMR (DMSO-d₆) of the title compound: δ 13.3 (s,1H), 10.7 (s, 1H), 10.0 (s, 1H), 7.90 (s, 1H), 7.82 (d, 1H, J=7.6), 7.69(d, 1H, J=8.4), 7.58 (m, 2H), 7.07 (d, 1H, J=8.4), 6.03 (s, 1H), 2.05(s, 4H, Acetone). HPLC: 97.6% purity at 26.08 min.

An X-ray powder diffractogram of the title composition is provided inFIG. 1. A differential scanning calorimetry curve of the titlecomposition is provided in FIG. 2, along with a thermal gravimetricanalysis curve. Tabulated characteristics of the X-ray powderdiffractogram in FIG. 1 are provided below in Table 1, which listsdiffraction angle 2θ, inter-planar distances d, and relative intensity(expressed as a percentage with respect to the most intense peak).

TABLE 1 X-RAY POWDER DIFFRACTOGRAM DATA. Angle [2θ] d-spacing [Å]Relative Intensity [%] 6.2 14.3 100 7.2 12.3 14.4 8.3 10.7 54.6 14.7 6.032.6 15.7 5.6 12.6 15.8 5.6 16.2 16.8 5.3 13.5 17.5 5.1 39.7 17.9 5.020.7 18.5 4.8 23.3 19.0 4.7 23.0 19.6 4.5 11.4 20.1 4.4 18.1 20.2 4.423.5 20.4 4.3 10.8 20.9 4.3 31.0 21.9 4.1 11.9 23.5 3.8 20.2 23.7 3.713.4 24.1 3.7 12.2 24.4 3.7 21.0 24.6 3.6 16.0 25.3 3.5 13.4 25.9 3.460.9 26.5 3.4 12.9 27.9 3.2 10.9 28.1 3.2 11.9 29.1 3.1 14.0

Example 3 Preparation of Crystalline Form I of3-Chloro-N-(((3-Chloro-5-Fluorophenyl)Amino)((5-(Trifluoromethyl)-1H-Pyrazol-3-Yl)Amino)Methylene)Benzamide

3-Chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamide,acetone solvate (16 g) was dried (60° C./10 Torr) to afford3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamideas a crystalline solid designated herein as Form I. Analytical analysisdid not identify solvate molecules in the crystalline solid. ¹H NMR(DMSO-d₆) of the title compound: δ 13.3 (s, 1H), 10.7 (s, 1H), 10.0 (s,1H), 7.90 (s, 1H), 7.82 (d, 1H, J=7.6), 7.69 (d, 1H, J=8.4), 7.58 (m,2H), 7.07 (d, 1H, J=8.4), 6.03 (s, 1H). HPLC: 97.6% purity at 26.08 min.

An X-ray powder diffractogram of the title composition is provided inFIG. 3. A differential scanning calorimetry curve of the titlecomposition is provided in FIG. 4, along with a thermal gravimetricanalysis curve. Tabulated characteristics of the X-ray powderdiffractogram in FIG. 3 are provided below in Table 2, which listsdiffraction angle 2θ, inter-planar distances d, and relative intensity(expressed as a percentage with respect to the most intense peak).

TABLE 2 X-RAY POWDER DIFFRACTOGRAM DATA. Angle [2θ] d-spacing [Å]Relative Intensity [%] 6.2 14.3 53.8 7.1 12.4 69.6 9.4 9.4 53.8 10.7 8.351.0 12.9 6.9 34.6 15.6 5.7 92.8 16.4 5.4 29.1 17.9 4.9 44.0 19.0 4.7100.0 20.0 4.4 62.6 21.9 4.1 38.9 23.6 3.8 51.4 25.2 3.5 57.8 27.3 3.324.0 28.4 3.1 15.8 35.0 2.6 10.0

Example 4 Preparation of Crystalline Form II of3-Chloro-N-(((3-Chloro-5-Fluorophenyl)Amino)((5-(Trifluoromethyl)-1H-Pyrazol-3-Yl)Amino)Methylene)Benzamide

3-Chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamide,acetone solvate (0.500 g) was dissolved in EtOH (5 mL) and warmed to 40°C. The mixture was clarified by filtration through celite and thendiluted with EtOH/water (1/1 by volume, 1.25 mL) at 40° C. Thesuspension was further diluted with EtOH/water (1/1 by volume, 5 mL) at40° C., and then the mixture was allowed to cool to room temperature andstirring was continued for 4 days. The resulting suspension wasfiltered, rinsed with EtOH/water (1/1 by volume, 5 mL), and dried (60°C./10 Torr) to afford3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamideas a crystalline solid designated herein as Form II. Analytical analysisdid not identify solvate molecules in the crystalline solid.

An X-ray powder diffractogram of the title composition is provided inFIG. 5. A differential scanning calorimetry curve of the titlecomposition is provided in FIG. 6. Tabulated characteristics of theX-ray powder diffractogram in FIG. 5 are provided below in Table 3,which lists diffraction angle 2θ, inter-planar distances d, and relativeintensity (expressed as a percentage with respect to the most intensepeak).

TABLE 3 X-RAY POWDER DIFFRACTOGRAM DATA. Angle [2θ] d-spacing [Å]Relative Intensity [%] 6.33 13.95 65.8 7.32 12.06 100.0 9.73 9.08 14.811.03 8.01 55.9 12.76 6.93 18.3 13.27 6.67 9.7 14.75 6.00 9.3 16.07 5.5112.7 16.91 5.24 27.2 18.45 4.80 9.9 19.18 4.62 42.0 19.53 4.54 8.3 20.564.31 19.8 22.20 4.00 88.4 22.51 3.95 43.5 23.11 3.85 56.1 23.55 3.7729.0 25.65 3.47 48.8 25.96 3.43 100.0 26.75 3.33 26.2 28.03 3.18 52.629.03 3.07 44.7 29.51 3.02 29.3 30.45 2.93 20.0 31.49 2.84 17.8 31.822.81 20.4 32.27 2.77 29.9 33.15 2.70 16.8 34.18 2.62 12.9 35.66 2.5259.6 36.03 2.49 17.7 36.84 2.44 13.5 37.75 2.38 14.8 38.27 2.35 10.938.99 2.31 13.6 39.71 2.27 13.5 42.15 2.14 11.2 43.46 2.08 13.7 44.192.05 8.3 44.55 2.03 13.1 45.96 1.97 16.3 46.46 1.95 16.6 48.33 1.88 9.449.33 1.85 9.9 50.27 1.81 9.3 50.48 1.81 9.9 51.25 1.78 8.2 51.45 1.779.5 53.86 1.70 9.1 55.63 1.65 9.8 56.54 1.63 6.5 57.53 1.60 8.8 59.281.56 6.4

Example 5 Alternative Procedure for Preparing Crystalline Form II of3-Chloro-N-(((3-Chloro-5-Fluorophenyl)Amino)((5-(Trifluoromethyl)-1H-Pyrazol-3-Yl)Amino)Methylene)Benzamide

3-Chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamideacetone solvate (176 g) was dissolved in EtOH (875 mL) and warmed to 40°C. The mixture was clarified by filtration through celite and thendiluted with EtOH/water (1/1 mixture by volume, 175 mL) at 40° C. FormII crystals of3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamide(437 mg) were added to the mixture. Next, the suspension was furtherdiluted with EtOH/water (1/1 mixture by volume, 700 mL) at 40° C. over40 minutes, and then the mixture was allowed to cool to room temperatureand stirring was continued overnight. Finally, the resulting suspensionwas filtered, the isolated residue was rinsed with EtOH/water (1/1mixture by volume, 500 mL) and dried (60° C./10 Torr) to afford 130 g(74% recovery) of3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamideas crystalline solid Form II. Analytical analysis did not identifysolvate molecules in the crystalline solid.

An X-ray powder diffractogram of the title composition is provided inFIG. 7. A differential scanning calorimetry curve of the titlecomposition is provided in FIG. 8. Tabulated characteristics of theX-ray powder diffractogram in FIG. 7 are provided below in Table 4,which lists diffraction angle 2θ, inter-planar distances d, and relativeintensity (expressed as a percentage with respect to the most intensepeak).

TABLE 4 X-RAY POWDER DIFFRACTOGRAM DATA. Angle [2θ] d-spacing [Å]Relative Intensity [%] 6.34 13.94 70.2 7.32 12.06 100.0 9.71 9.10 23.611.01 8.03 66.5 12.74 6.95 26.9 13.25 6.68 16.6 14.71 6.02 15.8 16.055.52 20.3 16.88 5.25 39.6 18.43 4.81 16.1 19.17 4.63 63.4 19.52 4.5514.1 20.54 4.32 31.2 22.15 4.01 21.3 22.47 3.95 11.4 23.09 3.85 13.823.64 3.76 11.4 23.90 3.72 14.1 25.60 3.48 25.1 25.93 3.43 24.0 27.993.19 15.4 28.99 3.08 10.9 35.61 2.52 13.2

Example 6 Preparation of3-Chloro-N-(((3-Chloro-5-Fluorophenyl)Amino)((5-(Trifluoromethyl)-1H-Pyrazol-3-Yl)Amino)Methylene-¹³C)Benzamide-1,2,3,4,5,6-¹³C₆

The title compound was prepared according to the following procedures.

Part I: Preparation of 3-Chlorobenzoic-1,2,3,4,5,6-¹³C₆ Acid

To a solution of 1-chloro-3-iodobenzene-1,2,3,4,5,6-¹³C₆ (1 g, 4.1 mmol)in anhydrous tetrahydrofuran (12 mL) at −78° C. under a nitrogenatmosphere was added a 2.0M solution of isopropylmagnesium chloride (2.3mL, 4.5 mmol) in tetrahydrofuran dropwise over 5 minutes. The resultingmixture was stirred at a temperature of −78° C. for 10 minutes, thencarbon dioxide gas was bubbled into solution, keeping a steady stream ofcarbon dioxide bubbling throughout reaction. Next, the reaction mixturewas stirred at −78° C. for 15 minutes, then the reaction vessel wasremoved from the cooling bath. Once the reaction mixture warmed toambient temperature, the reaction solution was diluted with diethylether (40 mL) and product extracted into 2M sodium hydroxide (3×20 mL).The aqueous extracts were combined and cooled by placing a vessel in anice bath, and then the aqueous extracts in the cooled vessel wereacidified with 6M hydrogen chloride. The product was extracted from theaqueous extracts using diethyl ether (3×20 mL). The organic extractswere combined and washed with brine, dried with magnesium sulfate,filtered, and concentrated to provide the title compound (600 mg, 90%).ESI m/z 161.07, 163.04 (M-H)⁻.

Part II: Preparation of3-Chloro-N-((3-chloro-5-fluorophenyl)carbamothioyl-¹³C)benzamide-1,2,3,4,5,6-¹³C₆

To a suspension of 3-chlorobenzoic-1,2,3,4,5,6-¹³C₆ acid (0.44 g, 2.7mmol) in anhydrous dichloromethane (10 mL) at 0° C. under a nitrogenatmosphere was added oxalyl chloride (0.34 mL, 4.1 mmol), followed byaddition of anhydrous N,N-dimethylformamide (30 μL). Some gas evolutionnoted. The reaction vessel was removed from the cooling bath, and thereaction mixture was allowed to warm to ambient temperature. Suspendedsolids in the reaction mixture slowly dissolved. Next, the reactionmixture was stirred at ambient temperature for 2 hours. Then, volatileswere removed in vacuo. The resulting residue was azeotroped with toluene(3×), then with chloroform (3×). The resulting oil was dissolved inacetonitrile (8 mL) to provide a mixture that was cooled with an icewater bath. To the cooled solution was added potassium thiocyanate-¹³C(0.28 g, 2.8 mmol) in a single portion. After 15 minutes, the reactionvessel was removed from the cooling bath and the reaction mixture wasstirred at room temperature for 1 hour. To the resulting suspension wasadded 3-chloro-5-fluoroaniline (0.39 g, 2.7 mmol). After the addition ofthe aniline, the reaction mixture became very viscous and was dilutedwith acetonitrile (5 mL). The reaction mixture was stirred at ambienttemperature for 1 hour. Then, water (10 mL) was added which caused asolid to form. The solids were filtered off, and then the solids werewashed with water and dried in a vacuum oven at 50° C. overnight toprovide the title compound (820 mg, 86% yield).

Part III: Preparation of3-Chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene-¹³C)benzamide-1,2,3,4,5,6-¹³C₆

To a suspension of3-chloro-N-((3-chloro-5-fluorophenyl)carbamothioyl-¹³C)benzamide-1,2,3,4,5,6-¹³C₆(0.77 g, 2.2 mmol) in methyl t-butyl ether (5 mL) at 35° C. was added5-(trifluoromethyl)-1H-pyrazol-3-amine (0.4 g, 2.6 mmol) followed byN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (0.65 g,3.3 mmol). The resulting suspension was stirred for 3 hours at 35° C.,during which time the solids were periodically scraped down sides of thereaction vessel. Next, the reaction mixture was partitioned betweenmethyl t-butyl ether and brine, the organic layer was separated anddried with sodium sulfate, filtered through celite, then concentratedonto coarse silica. The mixture was purified by column chromatographyeluting with a gradient of 0% to 10% v/v ethyl acetate in hexanes toprovide the title compound (320 mg, 30% yield).

Example 7 Preparation of4-Chloro-N-((3-Chloro-5-Fluorophenyl)Carbamothioyl)Benzamide

4-Chlorobenzoyl chloride (10.2 g, 58.5 mmol) was dissolved inacetonitrile (230 mL) and the reaction vessel containing this mixturewas cooled in an ice/water bath. To this mixture was added potassiumthiocyanate (6.25 g, 64.4 mol, 1.10 equiv). The cooling bath was removedfrom the reaction vessel after 15 minutes and the resulting reactionslurry was stirred at room temperature for approximately 1 hour.3-Chloro-5-fluoroaniline (7.05 mL, 70.2 mol, 1.2 equiv) was added as asolution in acetonitrile (7 mL) over 5 minutes to the reaction mixture.Next, the reaction mixture was stirred at room temperature overnight andthen quenched with water (230 mL), and the resulting mixture was stirredat room temperature for 1 hour. The mixture was filtered to collect thesolids, the isolated solids were rinsed with acetonitrile/water (1/1mixture by volume, 50 mL), and the resulting solid was dried at 60° C.in vacuo to provide 20.5 g the title compound as a solid. ¹H NMR(DMSO-d₆) of the title compound: δ 12.5 (s, 1H), 11.8 (s, 1H), 8.01 (t,1H, J=1.8), 7.96 (d, 2H, J=8.6), 7.67 (m, 2H), 7.59 (d, 2H, J=8.6), 7.33(d, 1H, J=8.4). HPLC: >99% purity at 23.52 min.

Example 8 Preparation of4-Chloro-N-(((3-Chloro-5-Fluorophenyl)Amino)((5-(Trifluoromethyl)-1H-Pyrazol-3-Yl)Amino)Methylene)Benzamide

A suspension of4-chloro-N-((3-chloro-5-fluorophenyl)carbamothioyl)benzamide (20.5 g,59.7 mmol) in TBME (500 mL) was heated to 50° C.5-(Trifluoromethyl)-1H-pyrazol-3-amine (10.8 g, 71.7 mmol, 1.20 equiv)and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) (17.6 g, 89.6mmol, 1.50 equiv) were added. The resulting reaction mixture was stirredat 40° C. for 3 hr and then diluted with a saturated aqueous sodiumchloride solution (0.5 L). The organic layer of the resulting mixturewas isolated, dried (Na₂SO₄), filtered, and concentrated. The resultingresidue was diluted with EtOAc (20 mL), and this mixture was warmed to50° C., then diluted with heptane (20 mL). Next, the heat source wasremoved and the mixture was allowed to cool to room temperature, then anice/water bath was applied to the reaction vessel to affectprecipitation. The resulting solids were collected by filtration, rinsedwith EtOAc/heptane (1/1 mixture by volume, 20 mL) and air dried at roomtemperature. Then, the solids were dissolved in EtOH (80 mL) and theresulting mixture warmed to 40° C. EtOH/water (1/1 mixture by volume, 80mL) was added to the mixture slowly over 40 minutes. Then, the heatsource was removed from the reaction vessel and the reaction mixture wasallowed to stir at room temperature for 2 days. Then, the reactionmixture was filtered, collected solids were rinsed with EtOH/water (1/1mixture by volume, 15 mL), and dried (60° C./10 Torr) to afford thetitle compound (15.2. g, 57% yield). ¹H NMR (DMSO-d₆) of the titlecompound: δ 13.3 (s, 1H), 10.7 (s, 1H), 10.0 (s, 1H), 7.90 (s, 1H), 7.90(d, 2H, J=8.4), 7.72 (d, 1H, J=11.5), 7.63 (m, 3H), 7.07 (d, 1H, J=8.5),6.00 (s, 1H). HPLC: 98.6% purity at 26.02 min.

Example 9 Biological Activity Towards ATPase and Ramos Cells

Exemplary compounds were tested for activity against F₁F₀-ATPase andRamos cells. Experimental procedures and results are described below.

Part I

Compounds in Table 5 below were tested for activity against F₁F₀-ATPaseby measuring the ability of the compounds to inhibit ATP synthesis. Inaddition, the compounds were assessed for cytotoxicity in Ramos cells.Results of the biological activity tests are shown in Table 5 below.Inhibition of F₁F₀-ATPase activity in synthesizing ATP and cytotoxicityin Ramos cells were measured according to the procedures described in K.M. Johnson et al. Chemistry & Biology 2005, 12, 485-496.

TABLE 5 Compound ATP Syn Ramos Cell No. Compound Structure IC₅₀ (μM)EC₅₀ (μM) 1 Crystalline Form I of 3-chloro-N-(((3-chloro-5- 0.04 0.05fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamide 2

0.09 0.1 3

0.31 1.84 4

0.04 0.12

Part II

The activity of compound3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamidetowards F₁F₀-ATPase and Ramos cells was evaluated by performing multipletimes the assay procedures described in Part I. In particular, activityagainst F₁F₀-ATPase was evaluated by measuring the ability of thecompound to inhibit ATP synthesis in twelve independent experiments.Cytotoxicity towards Ramos cells was measured in thirteen independentexperiments. Consistent with the procedure in Part I, the compound wasdissolved in dimethylsulfoxide to form a solution that was applied toF₁F₀-ATPase or Ramos cells, and the assays were performed according toprocedures described in K. M. Johnson et al. Chemistry & Biology 2005,12, 485-496.

IC₅₀ values (for inhibition of F₁F₀-ATPase activity in synthesizing ATP)from multiple replications of the experimental procedure were averaged.The mean IC₅₀ value for3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamidefor inhibition of F₁F₀-ATPase activity in synthesizing ATP wasdetermined to be 27±1.7 nM.

Similarly, EC₅₀ values (for cytotoxicity towards Ramos cells) frommultiple replications of the experimental procedure were averaged. Themean EC₅₀ value for3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamidefor cytotoxicity towards Ramos cells was determined to be 59±6 nM.

Example 10 Inhibition of Cytochrome P450 2D6

Compounds in Table 6 below were tested for activity against CytochromeP450 2D6 (hereinafter “CYP2D6”). Procedures and results are describedbelow.

Part I—Test Procedure:

Master solutions were prepared containing human liver microsomes (Gibco,0.2 mg/mL) and MgCl₂ (5 mM) in potassium phosphate buffer (10 mM). Toaliquots (169 μL) of the microsome solution was added test compound inacetonitrile (1 μL) and DMSO (1 μL) to provide final test compoundconcentrations of 0, 0.005, 0.05, 0.25, 1, 5, 10, and 25 μM.

NADPH (10 mM) in ultra-pure water (20 μL) was added, and this mixturewas incubated at 37° C. for 30 minutes. The enzyme reaction then wasinitiated by the addition of enzyme substrate (dextromethorphan)dissolved in 1 μL of acetonitrile and 9 μL of ultra-pure water. Thefinal substrate concentration was 10 μM.

After 20 minutes, the incubation mixture was diluted with 3 volumes ofcold methanol containing imipramine (200 nM), labetalol (200 nM), andketoprofen (2 μM) as internal standards. Samples were centrifuged at16,000 g for 10 minutes, then an aliquot of the supernatant (200 μL) wasremoved and analyzed by LC/MS/MS.

LC/MS/MS analyses were performed in duplicate using a Shimadzu HPLC andan API 4000 mass spectrometer. Liquid chromatography employed aPhenomenex C18, 5 μm, 50×2 mm column fitted with a guard column. SolventA was acetonitrile containing 0.1% v/v formic acid. Solvent B was watercontaining 0.1% v/v formic acid. Elution was performed at 0.5 mL perminute: 0-2 min, gradient of 5% Solvent A/95% Solvent B to 100% SolventA; 2.0-2.2 min, 100% Solvent A; 2.2-2.4 min, gradient of 100% Solvent Ato 5% Solvent A/95% Solvent B; 2.4-3.0 min, Solvent 5% A/Solvent B 95%.

The formation of metabolites was evaluated by peak area, and data wereanalyzed using Prism 5.0 software (GraphPad). The amount of metaboliteformed compared to a control sample containing no test compound wasplotted as a function of test compound concentration.

The IC₅₀ value of the test compound was determined from the plot as theconcentration at which the test compound confers 50% inhibition ofmetabolite formation.

Part II—Results:

IC₅₀ inhibition values for Compound 1 and Compound 2 are provided inTable 6. Compound 1 was approximately three-fold less potent ininhibiting CYP2D6 compared to Compound 2 in the experiment.

TABLE 6 Inhibition of CYP2D6 Thirty Minutes After Administration of TestCompound. Compound CYP2D6 IC₅₀ No. Compound Structure (μM) 1 CrystallineForm I of 3-chloro-N-(((3-chloro-5- 3.2fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamide 2

1.1

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent documents and scientificarticles referred to herein is incorporated by reference for allpurposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

1. A compound represented by Formula I:

or a geometric isomer or tautomer; or a pharmaceutically acceptable saltor solvate of any of the foregoing; wherein R¹ is a chloro group locatedat either the meta-position or para-position of the phenyl group towhich it is attached.
 2. The compound of claim 1, wherein the compoundis a compound of Formula I or a geometric isomer or tautomer, or apharmaceutically acceptable salt of any of the foregoing.
 3. Thecompound of claim 1, wherein the compound is in the form of a solvate.4. The compound of claim 1, wherein the compound is represented by:

or a geometric isomer or tautomer; or a pharmaceutically acceptable saltof any of the foregoing.
 5. The compound of claim 1, wherein thecompound is represented by:

or a geometric isomer or tautomer; or a solvate of any of the foregoing.6. The compound of claim 5, wherein the compound is in the form of anacetone solvate.
 7. The compound of claim 1, wherein the compound isrepresented by:

or a geometric isomer or tautomer.
 8. The compound of claim 1, whereinthe compound is represented by:


9. Crystalline compound3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamide.10. The crystalline compound of claim 9, wherein the crystallinecompound exhibits an X-ray powder diffraction pattern comprising peaksat the following diffraction angles (2θ): 6.2±0.2, 7.1±0.2, 9.4±0.2,10.7±0.2, 15.6±0.2, 19.0±0.2, 20.0±0.2, and 25.2±0.2.
 11. Thecrystalline compound of claim 10, wherein the relative intensity of thepeak at said diffraction angles (2θ) is at least 30%.
 12. Thecrystalline compound of claim 10 characterized by the following X-raypowder diffraction pattern expressed in terms of diffraction angle 2θ,inter-planar distances d, and relative intensity (expressed as apercentage with respect to the most intense peak): Angle [2θ] d-spacing[Å] Relative Intensity [%] 6.2 14.3 53.8 7.1 12.4 69.6 9.4 9.4 53.8 10.78.3 51.0 12.9 6.9 34.6 15.6 5.7 92.8 16.4 5.4 29.1 17.9 4.9 44.0 19.04.7 100.0 20.0 4.4 62.6 21.9 4.1 38.9 23.6 3.8 51.4 25.2 3.5 57.8 27.33.3 24.0 28.4 3.1 15.8 35.0 2.6 10.0


13. The crystalline compound of claim 10, wherein the X-ray powderdiffraction pattern is substantially as shown in FIG.
 3. 14. Thecrystalline compound of claim 11, wherein the crystalline compound has amelting point onset as determined by differential scanning calorimetryin the range of from about 157 degrees Celsius to about 161 degreesCelsius.
 15. The crystalline compound of claim 11, wherein thecrystalline compound has a melting point onset as determined bydifferential scanning calorimetry at about 159 degrees Celsius.
 16. Thecrystalline compound of claim 11, wherein the crystalline compound has adifferential scanning calorimetry curve substantially the same as shownin FIG.
 4. 17. The crystalline compound of claim 9, wherein thecrystalline compound exhibits an X-ray powder diffraction patterncomprising peaks at the following diffraction angles (2θ): 6.3±0.2,7.3±0.2, 11.0±0.2, 12.8±0.2, 16.9±0.2, 19.2±0.2, 20.6±0.2, 22.2±0.2,25.7±0.2, 26.0±0.2, and 35.7±0.2.
 18. The crystalline compound of claim17, wherein the relative intensity of the peak at said diffractionangles (2θ) is at least 15%.
 19. The crystalline compound of claim 17characterized by the following X-ray powder diffraction patternexpressed in terms of diffraction angle 2θ, inter-planar distances d,and relative intensity (expressed as a percentage with respect to themost intense peak): Angle [2θ] d-spacing [Å] Relative Intensity [%] 6.3313.95 65.8 7.32 12.06 100.0 9.73 9.08 14.8 11.03 8.01 55.9 12.76 6.9318.3 13.27 6.67 9.7 14.75 6.00 9.3 16.07 5.51 12.7 16.91 5.24 27.2 18.454.80 9.9 19.18 4.62 42.0 19.53 4.54 8.3 20.56 4.31 19.8 22.20 4.00 88.422.51 3.95 43.5 23.11 3.85 56.1 23.55 3.77 29.0 25.65 3.47 48.8 25.963.43 100.0 26.75 3.33 26.2 28.03 3.18 52.6 29.03 3.07 44.7 29.51 3.0229.3 30.45 2.93 20.0 31.49 2.84 17.8 31.82 2.81 20.4 32.27 2.77 29.933.15 2.70 16.8 34.18 2.62 12.9 35.66 2.52 59.6 36.03 2.49 17.7 36.842.44 13.5 37.75 2.38 14.8 38.27 2.35 10.9 38.99 2.31 13.6 39.71 2.2713.5 42.15 2.14 11.2 43.46 2.08 13.7 44.19 2.05 8.3 44.55 2.03 13.145.96 1.97 16.3 46.46 1.95 16.6 48.33 1.88 9.4 49.33 1.85 9.9 50.27 1.819.3 50.48 1.81 9.9 51.25 1.78 8.2 51.45 1.77 9.5 53.86 1.70 9.1 55.631.65 9.8 56.54 1.63 6.5 57.53 1.60 8.8 59.28 1.56 6.4


20. The crystalline compound of claim 17, wherein the X-ray powderdiffraction pattern is substantially as shown in FIG.
 5. 21. Thecrystalline compound of claim 18, wherein the crystalline compound has amelting point onset as determined by differential scanning calorimetryin the range of from about 158 degrees Celsius to about 165 degreesCelsius.
 22. The crystalline compound of claim 18, wherein thecrystalline compound has a differential scanning calorimetry curvesubstantially the same as shown in FIG.
 6. 23. The crystalline compound3-chloro-N-(((3-chloro-5-fluorophenyl)amino)((5-(trifluoromethyl)-1H-pyrazol-3-yl)amino)methylene)benzamideacetone solvate exhibiting an X-ray powder diffraction patterncomprising peaks at the following diffraction angles (2θ): 6.2±0.2,8.3±0.2, 14.7±0.2, 17.5±0.2, 17.9±0.2, 18.5±0.2, 20.2±0.2, 20.9±0.2,23.5±0.2, and 25.9±0.2.
 24. The crystalline compound of claim 23,wherein the relative intensity of the peak at said diffraction angles(2θ) is at least 15%.
 25. The crystalline compound of claim 23characterized by the following X-ray powder diffraction patternexpressed in terms of diffraction angle 2θ, inter-planar distances d,and relative intensity (expressed as a percentage with respect to themost intense peak): Angle [2θ] d-spacing [Å] Relative Intensity [%] 6.214.3 100 7.2 12.3 14.4 8.3 10.7 54.6 14.7 6.0 32.6 15.7 5.6 12.6 15.85.6 16.2 16.8 5.3 13.5 17.5 5.1 39.7 17.9 5.0 20.7 18.5 4.8 23.3 19.04.7 23.0 19.6 4.5 11.4 20.1 4.4 18.1 20.2 4.4 23.5 20.4 4.3 10.8 20.94.3 31.0 21.9 4.1 11.9 23.5 3.8 20.2 23.7 3.7 13.4 24.1 3.7 12.2 24.43.7 21.0 24.6 3.6 16.0 25.3 3.5 13.4 25.9 3.4 60.9 26.5 3.4 12.9 27.93.2 10.9 28.1 3.2 11.9 29.1 3.1 14.0


26. The crystalline compound of claim 23, wherein the X-ray powderdiffraction pattern is substantially as shown in FIG.
 1. 27. Thecrystalline compound of claim 24, wherein the crystalline compound has amelting point onset as determined by differential scanning calorimetryat about 159 degrees Celsius.
 28. The crystalline compound claim 24,wherein the crystalline compound has a differential scanning calorimetrycurve substantially the same as shown in FIG.
 2. 29. A pharmaceuticalcomposition comprising a compound of claim 1 and a pharmaceuticallyacceptable carrier.
 30. A pharmaceutical composition comprising acompound of claim 4 and a pharmaceutically acceptable carrier.
 31. Apharmaceutical composition comprising a compound of claim 5 and apharmaceutically acceptable carrier.
 32. A pharmaceutical compositioncomprising a compound of claim 7 and a pharmaceutically acceptablecarrier.
 33. A pharmaceutical composition comprising a crystallinecompound of claim 9 and a pharmaceutically acceptable carrier.
 34. Apharmaceutical composition comprising a crystalline compound of claim 10and a pharmaceutically acceptable carrier.
 35. A pharmaceuticalcomposition comprising a crystalline compound of claim 17 and apharmaceutically acceptable carrier.
 36. A pharmaceutical compositioncomprising a crystalline compound of claim 18 and a pharmaceuticallyacceptable carrier.
 37. A method of treating a disorder selected fromthe group consisting of an immune disorder, inflammatory disorder,cardiovascular disease, myeloma, lymphoma, cancer, and bacterialinfection, comprising administering to a patient in need thereof atherapeutically effective amount of a compound of claim 1 in order toameliorate a symptom of the disorder.
 38. The method of claim 37,wherein the disorder is rheumatoid arthritis, psoriasis, chronicgraft-versus-host disease, acute graft-versus-host disease, Crohn'sdisease, inflammatory bowel disease, multiple sclerosis, systemic lupuserythematosus, Celiac Sprue, idiopathic thrombocytopenic thromboticpurpura, myasthenia gravis, Sjogren's syndrome, scleroderma, ulcerativecolitis, asthma, uveitis, or epidermal hyperplasia.
 39. The method ofclaim 37, wherein the disorder is Crohn's disease or ulcerative colitis.40. The method of claim 37, wherein the disorder is cartilageinflammation, bone degradation, arthritis, juvenile arthritis, juvenilerheumatoid arthritis, pauciarticular juvenile rheumatoid arthritis,polyarticular juvenile rheumatoid arthritis, systemic onset juvenilerheumatoid arthritis, juvenile ankylosing spondylitis, juvenileenteropathic arthritis, juvenile reactive arthritis, juvenile Reter'sSyndrome, SEA Syndrome, juvenile dermatomyositis, juvenile psoriaticarthritis, juvenile scleroderma, juvenile systemic lupus erythematosus,juvenile vasculitis, pauciarticular rheumatoid arthritis, polyarticularrheumatoid arthritis, systemic onset rheumatoid arthritis, ankylosingspondylitis, enteropathic arthritis, reactive arthritis, Reter'sSyndrome, dermatomyositis, psoriatic arthritis, vasculitis, myolitis,polymyolitis, dermatomyolitis, osteoarthritis, polyarteritis nodossa,Wegener's granulomatosis, arteritis, polymyalgia rheumatica,sarcoidosis, sclerosis, primary biliary sclerosis, sclerosingcholangitis, dermatitis, atopic dermatitis, atherosclerosis, Still'sdisease, chronic obstructive pulmonary disease, Guillain-Barre disease,Type I diabetes mellitus, Graves' disease, Addison's disease, Raynaud'sphenomenon, or autoimmune hepatitis.
 41. The method of claim 37, whereinthe patient is a human.
 42. A method of inhibiting a F₁F₀-ATPase,comprising exposing a F₁F₀-ATPase to a compound of claim 1 to inhibitsaid F₁F₀-ATPase.
 43. The method of claim 42, wherein the F₁F₀-ATPase ismitochondrial F₁F₀-ATPase.
 44. The method of claim 37, wherein thecompound is a compound of claim
 4. 45. The method of claim 37, whereinthe compound is a compound of claim
 7. 46. The method of claim 37,wherein the compound is a compound of claim
 9. 47. The method of claim37, wherein the compound is a compound of claim
 10. 48. The method ofclaim 37, wherein the compound is a compound of claim 17.